DK2291523T3 - Factor VIII, von Willebrand factor or complexes thereof with prolonged in vivo half-life - Google Patents

Description

Description

Field of the invention: [0001] The present invention relates to modified nucleic acid sequences for von Willebrand factor (VWF) as well as complexes thereof and their derivatives, recombinant expression vectors containing such nucleic acid sequences, host cells transformed with such recombinant expression vectors, recombinant polypeptides and derivatives coded for by said nucleic acid sequences which recombinant polypeptides and derivatives do have biological activities together with prolonged in vivo half-life and/or improved in vivo recovery compared to the unmodified wild-type protein. The present invention further relates to processes for the manufacture of such recombinant proteins and their derivatives. The invention also relates to a transfer vector for use in human gene therapy, which comprises such nucleic acid sequences.

Background of the invention: [0002] There are various bleeding disorders caused by deficiencies of blood coagulation factors. The most common disorders are hemophilia A and B, resulting from deficiencies of blood coagulation factor VIII and IX, respectively. Another known bleeding disorder is von Willebrand’s disease.

[0003] In plasma FVIII exists mostly as a noncovalent complex with VWF and its coagulant function is to accelerate factor IXa dependent conversion of factor X to Xa Due to the complex formation of FVIII and VWF it was assumed for a long time that FVIII and VWF functions are two functions of the same molecule. Only in the seventies it became clear that FVIII and VWF are separate molecules that form a complex under physiologic conditions. In the eighties then the dissociation constant of about 0.2 nmol/L was determined (Leyte et al., Biochem J 1989, 257: 679-683) and the DNA sequence of both molecules was studied.

[0004] Classic hemophilia or hemophilia A is an inherited bleeding disorder. It results from a chromosome X-linked deficiency of blood coagulation FVIII, and affects almost exclusively males with an incidence of between one and two individuals per 10.000. The X-chromosome defect is transmitted by female carriers who are not themselves hemophiliacs. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with FVIII concentrates was introduced the mean life span for a person with severe hemophilia was less than 20 years. The use of concentrates of FVIII from plasma has considerably improved the situation for the hemophilia A patients increasing the mean lifespan extensively, giving most of them the possibility to live a more or less normal life. However, there have been certain problems with the plasma derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, viruses causing hepatitis B, non-A non-B hepatitis and AIDS have hit the population seriously. Since then different virus inactivation methods and new highly purified FVIII concentrates have recently been developed which established a very high safety standard also for plasma derived FVIII.

[0005] The cloning ofthe cDNAfor FVIII (Wood etal. 1984. Nature 312:330-336; Veharetal. 1984. Nature 312:337-342) made it possible to express FVIII recombinantly leading to the development of several recombinant FVIII products, which were approved by the regulatory authorities between 1992 and 2003. The fact that the central B domain of the FVIII polypeptide chain residing between amino acids Arg-740 and Glu-1649 does not seem to be necessary for full biological activity has also led to the development of a B domain deleted FVIII.

[0006] The mature FVIII molecule consists of 2332 amino acids which can be grouped into three homologous A domains, two homologous C domains and a B Domain which are arranged in the order: A1-A2-B-A3-C1-C2. The complete amino acid sequence of mature human FVIII is shown in SEQ ID NO:15. During its secretion into plasma FVIII is processed intracellularly into a series of metal-ion linked heterodimers as single chain FVIII is cleaved at the B-A3 boundary and at different sites within the B-domain. This processing leads to heterogeneous heavy chain molecules consisting of the A1, the A2 and various parts of the B-domain which have a molecular size ranging from 90 kDa to 200 kDa. The heavy chains are bound via a metal ion to the light chains, which consist ofthe A3, the C1 and the C2 domain (Saenko et al. 2002. Vox Sang. 83:89-96). In plasma this heterodimeric FVIII binds with high affinity to von Willebrand Factor (VWF), which protects it from premature catabolism. The half-life of non-activated FVIII bound to VWF is about 12 hours in plasma.

[0007] Coagulation FVIII is activated via proteolytic cleavage by FXa and thrombin at amino acids Arg372 and Arg740 within the heavy chain and at Arg1689 in the light chain resulting in the release of von Willebrand Factor and generating the activated FVIII heterotrimerwhich will form the tenase complex on phospholipid surfaces with FIXa and FX provided that Ca2+ is present. The heterotrimer consists ofthe A1 domain, a 50 kDa fragment, the A2 domain, a 43 kDa fragment and the light chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of FVIII (FVIIIa) consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit relatively loosely associated with the A1 and the A3 domain.

[0008] To avoid excessive coagulation, FVIIIa must be inactivated soon after activation. The inactivation of FVIIIa via activated Protein C (APC) by cleavage at Arg336 and Arg562 is not considered to be the major rate-limiting step. It is rather the dissociation of the non covalently attached A2 subunit from the heterotrimer which is thought to be the rate limiting step in FVIIIa inactivation after thrombin activation (Fay et al. 1991. J. Biol. Chem. 266 8957, Fay & Smudzin 1992. J. Biol. Chem. 267:13246-50). This is a rapid process, which explains the short half-life of FVIIIa in plasma, which is only 2.1 minutes (Saenko et al. 2002. Vox Sang. 83:89-96).

[0009] In severe hemophilia A patients undergoing prophylactic treatment FVIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half-life of FVIII of about 12 to 14 hours. Each i.v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done at home by the patients themselves or by the parents of children being diagnosed for hemophilia A.

[0010] It would thus be highly desirable to create a FVIII with increased functional half-life allowing the manufacturing of pharmaceutical compositions containing FVIII, which have to be administered less frequently.

[0011] Several attempts have been made to prolong the half-life of non-activated FVIII either by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching polymers to FVIII (WO 94/15625, WO 97/11957 and US 4970300), by encapsulation of FVIII (WO 99/55306), by introduction of novel metal binding sites (WO 97/03193), by covalently attaching the A2 domain to the A3 domain either by peptidic (WO 97/40145 and WO 03/087355) or disulfide linkage (WO 02/103024A2) or by covalently attaching the A1 domain to the A2 domain (W02006/108590).

[0012] Another approach to enhance the functional half-life of FVIIIorVWFisby PEGylation of FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923) or by PEGylation of VWF (WO 2006/071801) which pegylated VWF by having an increased half-life would indirectly also enhance the half-life of FVIII present in plasma.

[0013] VWF, which is missing, functionally defect or only available in reduced quantity in different forms of von Wille-brand disease (VWD), is a multimeric adhesive glycoprotein present in the plasma of mammals, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of wild-type VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in mature plasma VWF (Fischer etal., FEBS Lett. 351: 345-348, 1994). After cleavage of the signal peptide in the endoplasmatic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 N-linked and 10 O-linked carbohydrate side chains are added. More important, VWF dimers are multimerized via N-terminal disulfide bridges and the propeptide of 741 amino acids length is cleaved off by the enzyme PACE/furin in the late Golgi apparatus. The propeptide as well as the high-molecular-weight multimers of VWF (VWF-HMWM) are stored in the Weibel-Pallade bodies of endothelial cells or in the α-Granules of platelets.

[0014] Once secreted into plasma the protease ADAMTS13 cleaves VWF within the A1 domain of VWF. Plasma VWF therefore consists of a whole range of multimers ranging from single dimers of 500 kDa to multimers consisting of up to more than 20 dimers of a molecular weight of over 10,000 kDa. The VWF-FIMWM hereby having the strongest hemostatic activity, which can be measured in ristocetin cofactor activity (VWF: RCo). The higher the ratio of VWF:RCo/VWF antigen, the higher the relative amount of high molecular weight multimers.

[0015] Defects in VWF are causal to von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms some of them being associated with the loss or the decrease of high molecular weight multimers. Von VWD type 2a is characterized by a loss of both intermediate and large multimers. VWD type 2B is characterized by a loss of highest-molecular-weight multimers. VWD is the most frequent inherited bleeding disorder in humans and can be treated by replacement therapy with concentrates containing VWF of plasmatic or recombinant origin. VWF can be prepared from human plasma as for example described in EP 05503991. EP 0784632 describes a method for isolating recombinant VWF.

[0016] In plasma FVIII binds with high affinity to von VWF, which protects it from premature catabolism and thus, plays in addition to its role in primary hemostasis a crucial role to regulate plasma levels of FVIII and as a consequence is also a central factor to control secondary hemostasis. The half-life of non-activated FVIII bound to VWF is about 12 to 14 hours in plasma. In von Willebrand disease type 3, where no or almost no VWF is present, the half-life of FVIII is only about 6 hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The stabilizing effect of VWF on FVIII has also been used to aid recombinant expression of FVIII in CHO cells (Kaufman et al. 1989, Mol Cell Biol).

[0017] Until today the standard treatment of Hemophilia A and VWD involves frequent intravenous infusions of preparations of FVIII and VWF concentrates or of concentrates comprising a complex of FVIII and VWF derived from the plasmas of human donors or in case of FVIII that of pharmaceutical preparations based on recombinant FVIII. While these replacement therapies are generally effective, e.g. in severe hemophilia A patients undergoing prophylactic treat- ment FVIII has to be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of FVIII of about 12 hours. Already above levels of 1% of the FVIII activity in non-hemophiliacs, e.g. by a raise of FVIII levels by 0. 01 U/ml, severe hemophilia A is turned into moderate hemophilia A. In prophylactic therapy dosing regimes are designed such that the trough levels of FVIII activity do not fall below levels of 2-3% of the FVIII activity in non-hemophiliacs. Each 1. v. administration is cumbersome, associated with pain and entails the risk of an infection especially as this is mostly done in home treatment by the patients themselves or by the parents of children being diagnosed for hemophilia A. In addition the frequent i.v. injections inevitably result in scar formation, interfering with future infusions. As prophylactic treatment in severe hemophilia is started early in life, with children often being less than 2 years old, it is even more difficult to inject FVIII 3 times per week into the veins of such small patients. For a limited period, implantation of port systems mayofFeran alternative. Despite the fact that repeated infections may occur and ports can cause inconvenience during physical exercise, they are nevertheless typically considered as favorable as compared to intravenous injections.

[0018] The in vivo half-life of human VWF in the human circulation is approximately 12 to 20 hours. In prophylactic treatment of VWD e.g. of type 3 it would also be highly desirable to find ways to prolong the functional half-life of VWF.

[0019] Another approach to enhance thefunctional half-life of VWF is by PEGylation (WO 2006/071801) which pegylat-ed VWF by having an increased half-life would indirectly also enhance the half-life of FVIII present in plasma.

[0020] Flowever the chemical conjugation of PEG or other molecules to therapeutic proteins always entails the risk, that the specific activity is reduced due to shielding of important interaction sites with other proteins, chemical conjugation adds an additional step in the manufacture of such proteins decreasing final yields and making manufacture more expensive. Also the long term effects on human health are not known as currently known PEGylated therapeutic proteins do not need to be administrated lifelong as it would be the case for a VWF to be administered in prophylaxis of von Willebrand disease or in for a FVIII to be administered in hemophilia A.

[0021] It would thus be highly desirable to obtain a long-lived VWF which is not chemically modified.

[0022] In the prior art fusions of coagulation factors to albumin (WO 01/79271), alpha-fetoprotein (WO 2005/024044) and immunoglobulin (WO 2004/101740) as half-life enhancing polypeptides have been described. These were taught to be attached to the carboxy- or the amino-terminus or to both termini of the respective therapeutic protein moiety, occasionally linked by peptidic linkers, preferably by linkers consisting of glycine and serine.

[0023] Ballance et al. (WO 01/79271) described N- or C-terminal fusion polypeptides of a multitude of different therapeutic polypeptides fused to human serum albumin. Long lists of potential fusion partners are described without disclosing experimental data for almost any of these polypeptides whether or not the respective albumin fusion proteins actually retain biological activity and have improved properties. Among said list of therapeutic polypeptides also FVIII and VWF are mentioned.

[0024] A man skilled in the art would not have considered fusing human albumin to the N- or the C-terminus of VWF. In an N-terminal fusion the albumin part would be cleaved off during propeptide processing. Or if the propeptide would be omitted the multimerization would not take place. As detailed above the C-terminus of VWF is essential for the initial dimerization and secretion as shown by Schneppenheim et al. (Schneppenheim R. et al. 1996. Defective dimerization of VWF subunits due to a Cys to Arg mutation in VWD type IID. Proc Natl Acad Sci USA 93:3581-3586; Schneppenheim R. et al. 2001. Expression and characterization of VWF dimerization defects in different types of VWD. Blood 97:2059-2066.), Baronciani et al. (Baronciani L.et al. 2000. Molecular characterization of a multiethnic group of 21 patients with VWD type 3. Thromb. Haemost 84:536-540), Enayat et al. (Enayat MS et al. 2001. Aberrant dimerization of VWF as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) VWD. Blood 98:674-680) and Tjernberg et al. 2006. Homozygous C2362F VWF induces intracellular retention of mutant VWF resulting in autosomal recessive severe VWD. BrJ Haematol. 133:409-418). Therefore the man skilled in the art would not consider fusing a large protein like human albumin to the C- or N-terminus of VWF as he would expect that normal dimerization or multimerization of VWF would be impaired. As the higher multimers of VWF are the ones most active in primary hemostasis the man skilled in the art would have looked for other ways to prolong the functional half-life of VWF.

[0025] It was now surprisingly found that fusing albumin to the C-terminal part of VWF, not only permits expression and secretion of VWF chimeric proteins from mammalian cells but also results in modified VWF molecules that retain significant VWF activity and form high molecular weight multimers. In addition, such modified VWF molecules exhibit prolonged in vivo half-life and/or improved in vivo recovery.

Description of the invention [0026] It is an objective of this invention to provide a modified VWF as well as complexes of non-modified FVIII with modified VWF with enhanced in vivo half-life.

[0027] The term "modified VWF" in the sense of the invention means VWF polypeptides which are fused to half-life enhancing polypeptides, encompassing also natural alleles, variants, deletions and insertions of VWF.

[0028] It is another objective of this invention to provide a modified VWF as well as complexes of complexes of non- modified FVIII with modified VWF with improved in vivo recovery.

[0029] Another objective of the invention is that this modified VWF as well as complexes of non-modified FVIII with modified VWF can be expressed by mammalian cells and retain their respective biological activities.

[0030] In summary, surprisingly the modified VWF as well as complexes of non-modified FVIII with modified VWF of the invention have retained biological activity, increased in vivo half-life and in vivo recovery.

[0031] The modified VWF as well as complexes of non-modified FVIII with modified VWF molecules of the invention can be generated by fusing a half-life enhancing protein (HLEP) moiety to the C-terminal part of FVIII or to the C-terminal part of VWF.

[0032] HLEPs in the sense of the present invention are selected from albumin.

[0033] The present invention therefore relates to modified VWF as well as complexes of non-modified FVIII with modified VWF having at the C-terminal part of the modified VWF a fusion to albumin, characterized in that the modified VWF or the complex of non-modified FVIII with modified VWF has prolonged functional half-life compared to the functional half-life of the wild-type VWF or the complex of wild-type VWF and wild-type FVIII..

[0034] Another aspect of the invention are polynucleotides or combinations of polynucleotides encoding the modified VWF.

[0035] The invention further relates to plasmids or vectors comprising a polynucleotide described herein, to host cells comprising a polynucleotide ora plasmid or vector described herein.

[0036] Another aspect of the invention is a method of producing a modified VWF or a complex of non-modified FVIII comprising: (a) culturing host cells of the invention under conditions such that the modified coagulation factor is expressed; and (b) optionally recovering the modified coagulation factor from the host cells or from the culture medium.

[0037] The invention further pertains to pharmaceutical compositions comprising a modified VWF or a complex of non-modified FVIII with modified VWF, a polynucleotide, ora plasmid or vector described herein.

[0038] Yet another aspect of the invention is the use of a modified VWF or a complex of non-modified FVIII with modified VWF, one or more polynucleotides, or one or more plasmids or vectors, or of host cells according to this invention for the manufacture of a medicament for the treatment or prevention of a blood coagulation disorder.

Detailed description of the invention [0039] The invention pertains to a modified VWF or a complex comprising non-modified FVIII and modified VWF wherein the modified VWF is fused at a C-terminal part of the primary translation polypeptide of VWF to the N-terminal part acid of albumin.

[0040] In preferred embodiments the invention pertains to a modified VWF or a complex comprising non-modified FVIII and modified VWF, wherein a. the modified VWF has a prolonged functional half-life compared to the functional half-life of wild-type VWF or b. the complex comprising non-modified FVIII and modified VWF has a prolonged functional half-life compared to the functional half-life of the corresponding complex comprising wild-type FVIII and wild-type VWF.

[0041] A preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide has a functional half-life increased by at least 25% as compared to the functional half-life of the corresponding wild-type polypeptide or the complex comprising said modified polypeptide or a complex comprising said modified polypeptides has a functional half-life increased by at least 25% as compared to the corresponding complex of wild-type FVIII and wild-type VWF.

[0042] Another embodiment of the invention is a modified VWF or a complex comprising non-modified FVIII and modified VWF, wherein a. the modified VWF has a prolonged antigen half-life compared to the antigen half-life of wild-type VWF or b. the complex comprising non-modified FVIII and modified VWF has a prolonged antigen half-life compared to the antigen half-life of the corresponding complex of wild-type FVIII and wild-type VWF.

[0043] A preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide has an antigen half-life increased by at least 25% as compared to the antigen half-life of the corresponding wild-type polypeptide or the complex comprising said modified polypeptide or a complex comprising said modified polypeptides has an antigen half-life increased by at least 25% as compared to the corresponding complex of wild-type FVIII and wild-type VWF.

[0044] Still another embodiment of the invention is a modified VWF or a complex comprising non-modified FVIII and modified VWF, wherein a. the modified VWF has an increased in vivo recovery compared to the in vivo recovery of wild-type VWF or b. the complex comprising non-modified FVIII and modified VWF has an increased in vivo recovery compared to the in vivo recovery of the corresponding complex comprising wild-type FVIII and wild-type VWF.

[0045] Another preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide has an in vivo recovery increased by at least 10% as compared to the in vivo recovery of the corresponding wild-type polypeptide or the complex comprising said modified polypeptide or a complex comprising said modified polypeptides has an in vivo recovery increased by at least 10% as compared to the corresponding complex of wild-type FVIII and wild-type VWF.

[0046] Another preferred embodiment of the invention is a) a modified polypeptide or a complex comprising said modified polypeptide as described above, wherein the vWF component of said complex is fused at the C-terminal amino acid of its primary translation product to the N-terminal part of albumin.

[0047] Another preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide as described above, wherein the modified polypeptide has at least 10% of the biological activity of wild-type polypeptide or the complex comprising the modified polypeptide has at least 10% of the biological activity of the corresponding complex of wild-type FVIII and wild-type VWF.

[0048] Also comprised in the present invention is a method of preparing a modified VWF having increased functional half-life, comprising fusing the N-terminal part of albumin to a C-terminal part of the primary translation polypeptide of the VWF as well as a method of preparing a complex comprising non-modified FVIII and modified VWF by mixing a wild-type FVIII with a modified VWF prepared by the method described above.

[0049] Also encompassed in the invention is the use of a. a wild-type FVIII and a modified VWF prepared by the method described above for the manufacture of a combined pharmaceutical preparation for simultaneous, separate or sequential use in the therapy of bleeding disorders, preferentially in the therapy of hemophilia A and/or von Willebrand disease.

[0050] The "functional half-life" according to the present invention is the half-life of the biological activity of the modified VWF or a complex of the non-modified FVIII with modified VWF once it has been administered to a mammal and can be measured in vitro in blood samples taken at different time intervals from said mammal after the modified VWF or the complex of non-modified FVIII with modified VWF has been administered.

[0051] The phrases "fusing" or "fused" refer to the addition of amino acids to the C-terminal part of VWF. When referring herein to a "fusion to the C-terminal amino acid of VWF" this means a fusion exactly to the C-terminal amino acid of VWF at amino acid 2050 of wild-type mature VWF. Mature VWF meaning the respective polypeptide after cleavage of the propeptide. However the invention also encompasses a "fusion to the C-terminal part of VWF" in the sense of this invention may also include a fusion to a VWF molecule in which one or more amino acid position up to n amino acids from the C-terminal amino acid of VWF are deleted. The figure n is an integer that should not be greater than 5%, preferably not greater than 1% of the total number of amino acids of the VWF. Usually, n is 20, preferably 15, more preferably 10, still more preferably 5 or less (e.g. 1,2, 3, 4 or 5).

[0052] In one embodiment the modified VWF has the following structure: [formula 2] wherein

N is an N-terminal part of VWF,

L1 is a chemical bond or a linker sequence H is albumin, and C is a C-terminal part of VWF

[0053] L1 may be a chemical bond or a linker sequence consisting of one or more amino acids, e.g. of 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3 (e.g. 1,2 or 3) amino acids and which may be equal or different from each other. Usually, the linker sequences are not present at the corresponding position in the wild-type coagulation factor. Examples of suitable amino acids present in L1 include Gly and Ser.

[0054] FVIII may be processed proteolytically at various stages. For example, as mentioned supra, during its secretion into plasma single chain FVIII is cleaved intracellularly at the B-A3 boundary and at different sites within the B-domain. The heavy chain is bound via a metal ion to the light chain having the domain structure A3-C1-C2. FVIII is activated via proteolytic cleavage at amino acids Arg372 and Arg740 within the heavy chain and at Arg1689 in the light chain generating the activated FVIII heterotrimer consisting of the A1 domain, the A2 domain, and the light chain (A3-C1-C2), a 73 kDa fragment. Thus the active form of FVIII (FVIIIa) consists of an A1-subunit associated through the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2 light chain and a free A2 subunit relatively loosely associated with the A1 and the A3 domain.

[0055] Preferably N - FVIII - C comprises the full length sequence of FVIII. Also encompassed are N-terminal, C-terminal or internal deletions of FVIII as long as the biological activity of FVIII is retained. The biological activity is retained in the sense of the invention if the FVIII with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type FVIII. The biological activity of FVIII can be determined by the artisan as described below.

[0056] Asuitable test to determine the biological activity of FVIII is for example the one stage or the two stage coagulation assay (Rizza etal. 1982. Coagulation assay of FVIILC and FIXain Bloom ed.The Hemophilias. NY Churchchill Livingston 1992) or the chromogenic substrate FVIILC assay (S. Rosen, 1984. Scand J Haematol 33:139-145, suppl.). The content of these references is incorporated herein by reference.

[0057] The cDNA sequence and the amino acid sequence of the mature wild-type form of human blood coagulation FVIII are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The reference to an amino acid position of a specific sequence means the position of said amino acid in the FVIII wild-type protein and does not exclude the presence of mutations, e.g. deletions, insertions and/or substitutions at other positions in the sequence referred to. For example, a mutation in "Glu2004" referring to SEQ ID NO:15 does not exclude that in the modified homologue one or more amino acids at positions 1 through 2332 of SEQ ID NO:15 are missing.

[0058] The terms "blood coagulation Factor VIII", "Factor VIII" and "FVIII" are used interchangeably herein. "Blood coagulation Factor VIII" includes wild-type blood coagulation FVIII as well as derivatives of wild-type blood coagulation FVIII having the procoagulant activity of wild-type blood coagulation FVIII. Derivatives may have deletions, insertions and/or additions compared with the amino acid sequence of wild-type FVIII. The term FVIII includes proteolytically processed forms of FVIII, e.g. the form before activation, comprising heavy chain and light chain.

[0059] The term "FVIII" includes any FVIII variants or mutants having at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type factor VIII.

[0060] As non-limiting examples, FVIII molecules include FVIII mutants preventing or reducing APC cleavage (Amano 1998. Thromb. Haemost. 79:557-563), FVIII mutants further stabilizing the A2 domain (WO 97/40145), FVIII mutants resulting in increased expression (Swaroop et al. 1997. JBC 272:24121-24124), FVIII mutants reducing its immuno-genicity (Lollar 1999. Thromb. Haemost. 82:505-508), FVIII reconstituted from differently expressed heavy and light chains (Oh et al. 1999. Exp. Mol. Med. 31:95-100), FVIII mutants reducing binding to receptors leading to catabolism of FVIII like HSPG (heparan sulfate proteoglycans) and/or LRP (low density lipoprotein receptor related protein) (Ananyeva et al. 2001. TCM, 11:251-257), disulfide bond-stabilized FVIII variants (Gale et al., 2006. J. Thromb. Hemost. 4:1315-1322), FVIII mutants with improved secretion properties (Miao etal., 2004. Blood 103:3412-3419), FVIII mutants with increased cofactor specific activity (Wakabayashi et al., 2005. Biochemistry 44:10298-304), FVIII mutants with improved biosynthesis and secretion, reduced ER chaperone interaction, improved ER-Golgi transport, increased activation or resistance to inactivation and improved half-life (summarized by Pipe 2004. Sem. Thromb. Hemost. 30:227-237). All of these FVIII mutants and variants are incorporated herein by reference in their entirety.

[0061] VWF may be processed proteolytically at various stages. For example, as mentioned supra, the protease ADAMTS13 cleaves VWF within the A2 domain of VWF. Accordingly, the present invention encompasses also modified VWF which has been cleaved proteolytically e.g. by ADAMTS13. Such cleavage would result in multimeric chains of VWF which comprise at their ends at least one or at most two monomers of VWF which have been cleaved byADAMTS 13.

[0062] Preferably N - VWF - C comprises the full length sequence of VWF. Also encompassed are N-terminal, C-terminal or internal deletions of VWF as long as the biological activity of VWF is retained. The biological activity is retained in the sense of the invention if the VWF with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type VWF. The biological activity of wild-type VWF can be determined by the artisan using methods for ristocetin cofactor activity (Federici AB et al. 2004. Haemato-logica 89:77-85), binding of VWF to GP Iba of the platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), ora collagen binding assay (Kallas & Talpsep. 2001. Annals of Hematology 80:466-471).

[0063] "FVIII" and/or "VWF" within the above definition also include natural allelic variations that may exist and occur from one individual to another. "FVIII" and/or "VWF" within the above definition further includes variants of FVIII and or VWF. Such variants differ in one or more amino acid residues from the wild-type sequence. Examples of such differences may include as conservative amino acid substitutions, i.e. substitutions within groups of amino acids with similar characteristics, e.g. (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, and (6) aromatic amino acids. Examples of such conservative substitutions are shown in the following table.

Table 1:

[0064] The functional half-life according to another embodiment of the invention is the half-life of the biological function of the VWF once it has been administered to a mammal and is measured in vitro. The functional half-life of the modified VWF according to the invention is greater than that of the VWF lacking the modification as tested in the same species. The functional half-life is increased by at least 10%, preferably increased by at least 25%, more preferably by at least 50%, and even more preferably by at least 100% compared to the VWF lacking the modification and/or to the wild-type form of VWF.

[0065] The functional half-life of a modified VWF comprising a FILEP modification, can be determined by administering the respective modified VWF (and in comparison that of the non-modified VWF) to rats, rabbits or other experimental animal species intravenously or subcutaneously and following the elimination of the biological activity of said modified or respectively non-modified VWF in blood samples drawn at appropriate intervals after application. Suitable test methods are the activity tests described herein.

[0066] As a surrogate marker for the half-life of biological activity also the levels of antigen of the wild-type FVIII or the levels of antigen of the modified or respectively wild-type VWF can be measured. Thus also encompassed by the invention are modified VWF molecules having at the C-terminal part of VWF a fusion to albumin, characterized in that the modified VWF or the complex of the non-modified FVIII with modified VWF has a prolonged half-life of the VWF antigen compared to the half-life of the VWF antigen lacking said insertion. The "half-life of the VWF antigen" according to the present invention is the half-life of the antigen of the VWF once it has been administered to a mammal and is measured in vitro. Antigen test methods based on specific antibodies in an enzyme immunoassay format as known to the artisan and commercially available (e.g. Dade Behring, Instrumentation Laboratory, Abbott Laboratories, Diagnostica Stago). Functional and antigen half-lives can be calculated using the time points ofthe beta phase of elimination according to the formula t1/2 = In2 / k, whereas k is the slope ofthe regression line.

[0067] In another embodiment ofthe invention, the modified VWF ofthe invention exhibits an improved in vivo recovery compared to the wild-type VWF. The in vivo recovery can be determined in vivo for example in models of VWD, like VWF knockout mice in which one would expect an increased percentage ofthe modified VWF ofthe invention be found by antigen or activity assays in the circulation shortly (5 to 10 min.) after i.v. administration compared to the corresponding wild-type VWF.

[0068] The in vivo recovery is preferably increased by at least 10%, more preferably by at least 20%, and even more preferably by at least 40% compared to wild-type VWF.

[0069] It is another objective of the present invention to provide long-lived VWF molecules, which after proteolytic processing in vivo do have functional properties comparable to that of an unmodified VWF. This can be achieved by maintaining or inserting certain cleavage sites in the modified VWF leading to a proteolytic cleavage for example when in contact with activated coagulation factors, which separates the VWF from the albumin. Accordingly, in one embodiment, the functional half-life ofthe proteolytically processed modified VWF is substantially the same as that ofthe non-modified VWF lacking the modification, and/or it is substantially the same as that ofthe wild-type VWF (e.g. ±15%, preferably ±10%).

[0070] Another preferred embodiment ofthe invention is a coexpression of wild-type VWF and a modified VWF according to the invention resulting in VWF multimers comprising non-modified as well as modified VWF monomers.

Linker sequences [0071] According to this invention, the therapeutic polypeptide moiety may be coupled to the albumin moiety by a peptide linker. The linker should be non-immunogenic and may be a non-cleavable or cleavable linker.

[0072] Non-cleavable linkers may be comprised of alternating glycine and serine residues as exemplified in W02007/090584.

[0073] In another embodiment of the invention the peptidic linker between the VWF moiety and the albumin moiety consists of peptide sequences, which serve as natural interdomain linkers in human proteins. Preferably such peptide sequences in their natural environment are located close to the protein surface and are accessible to the immune system so that one can assume a natural tolerance against this sequence. Examples are given in W02007/090584.

[0074] Cleavable linkers should be flexible enough to allow cleavage by proteases.

[0075] In a highly preferred embodiment the linker peptide is derived from FVIII itself and comprises of sequences encompassing the thrombin cleavage sites at amino acid positions 372, 740 and 1689 of SEQ ID NO. 15, respectively. In another preferred embodiment the linker peptide is derived from FX, FIX, FVII or FXI.

[0076] The linker peptides are preferably cleavable by the proteases of the coagulation system, for example Flla, FIXa, FXa, FXIa, FXI la and FVIIa.

Half-life enhancing polypeptides (HLEPs) [0077] A "half-life enhancing polypeptide" as used herein is selected from the group consisting of albumin.

Albumin as HLEP

[0078] The terms, "human serum albumin" (FISA) and "human albumin" (HA) and "albumin" (ALB) are used interchangeably in this application. The terms "albumin" and "serum albumin" are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

[0079] As used herein, "albumin" refers collectively to albumin polypeptide or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments thereof, especially the mature form of human albumin as shown in SEQ ID NO:16 herein or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

[0080] In particular, the proposed FVIII fusion and/or VWF fusion constructs of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. Generally speaking, an albumin fragment or variant will be at least 10, preferably at least 40, most preferably more than 70 amino acids long. The albumin variant may preferentially consist of or alternatively comprise at least one whole domain of albumin or fragments of said domains, for example domains 1 (amino acids 1-194 of SEQ ID NO:16), 2 (amino acids 195-387 of SEQ ID NO: 16), 3 (amino acids 388-585 of SEQ ID NO: 16), 1 + 2 (1-387 of SEQ ID NO: 16), 2 + 3 (195-585 of SEQ ID NO: 16) or 1 + 3 (amino acids 1-194 of SEQ ID NO: 16 + amino acids 388-585 of SEQ ID NO: 16). Each domain is itself made up of two homologous subdomains namely 1 -105,120-194,195-291,316-387,388-491 and 512-585, with flexible inter-subdomain linker regions comprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492 to Ala511.

[0081] The albumin portion of the proposed VWF fusion constructs of the invention may comprise at least one sub-domain or domain of HA or conservative modifications thereof.

Polynucleotides [0082] The invention furtherrelates to a polynucleotide encoding a modified VWF variant as described in this application. The term "polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNAor DNAor modified RNAorDNA. The polynucleotide may be single-or double-stranded DNA, single or double-stranded RNA. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs that comprise one or more modified bases and/or unusual bases, such as inosine. It will be appreciated that a variety of modifications may be made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.

[0083] The skilled person will understand that, due to the degeneracy of the genetic code, a given polypeptide can be encoded by different polynucleotides. These "variants" are encompassed by this invention.

[0084] Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term "isolated" polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified from a host cell. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

[0085] The invention further relates to a group of polynucleotides which together encode the modified VWF of the invention. A first polynucleotide in the group may encode the N-terminal part of the modified VWF, and a second polynucleotide may encode the C-terminal part of the modified VWF.

[0086] Yet another aspect of the invention is a plasmid or vector comprising a polynucleotide according to the invention. Preferably, the plasmid or vector is an expression vector. In a particular embodiment, the vector is a transfer vector for use in human gene therapy.

[0087] The invention also relates to a group of plasmids or vectors that comprise the above group of polynucleotides. A first plasmid or vector may contain said first polynucleotide, and a second plasmid or vector may contain said second polynucleotide.

[0088] Still another aspect of the invention is a host cell comprising a polynucleotide, a plasmid orvectorof the invention, or a group of polynucleotides or a group of plasmids or vectors as described herein.

[0089] The host cells of the invention may be employed in a method of producing a modified VWF, which is part of this invention. The method comprises: (a) culturing host cells of the invention under conditions such that the desired insertion protein is expressed; and (b) optionally recovering the desired insertion protein from the host cells or from the culture medium.

[0090] It is preferred to purify the modified VWF of the present invention to > 80% purity, more preferably > 95% purity, and particularly preferred is a pharmaceutically pure state that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, an isolated or purified modified VWF of the invention is substantially free of other, non-related polypeptides.

[0091] The various products of the invention are useful as medicaments. Accordingly, the invention relates to a pharmaceutical composition comprising a modified VWF as described herein, a polynucleotide of the invention, ora plasmid or vector of the invention.

[0092] The invention also concerns a method of treating an individual suffering from a blood coagulation disorder such as hemophilia A or B. The method comprises administering to said individual an efficient amount of the modified VWF or the complex of the non-modified FVIII with modified VWF as described herein. In another embodiment, the method comprises administering to the individual an efficient amount of a polynucleotide of the invention or of a plasmid or vector of the invention. Alternatively, the method may comprise administering to the individual an efficient amount of the host cells of the invention described herein.

Expression of the proposed mutants [0093] The production of recombinant mutant proteins at high levels in suitable host cells requires the assembly of the above-mentioned modified cDNAs into efficient transcriptional units together with suitable regulatory elements in a recombinant expression vector that can be propagated in various expression systems according to methods known to those skilled in the art. Efficient transcriptional regulatory elements could be derived from viruses having animal cells as their natural hosts or from the chromosomal DNA of animal cells. Preferably, promoter-enhancer combinations derived from the Simian Virus 40, adenovirus, BK polyoma virus, human cytomegalovirus, or the long terminal repeat of Rous sarcoma virus, or promoter-enhancer combinations including strongly constitutively transcribed genes in animal cells like beta-actin or GRP78 can be used. In order to achieve stable high levels of mRNA transcribed from the cDNAs, the transcriptional unit should contain in its 3’-proximal part a DNA region encoding a transcriptional termination-polyade-nylation sequence. Preferably, this sequence is derived from the Simian Virus 40 early transcriptional region, the rabbit beta-globin gene, or the human tissue plasminogen activator gene.

[0094] The cDNAs are then integrated into the genome of a suitable host cell line for expression of the VWF proteins. Preferably this cell line should be an animal cell-line of vertebrate origin in order to ensure correct folding, disulfide bond formation, asparagine-linked glycosylation and other post-translational modifications as well as secretion into the cultivation medium. Examples on other post-translational modifications are tyrosine O-sulfation and proteolytic processing of the nascent polypeptide chain. Examples of cell lines that can be use are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster CHO-cells.

[0095] The recombinant expression vector encoding the corresponding cDNAs can be introduced into an animal cell line in several different ways. For instance, recombinant expression vectors can be created from vectors based on different animal viruses. Examples of these are vectors based on baculovirus, vaccinia virus, adenovirus, and preferably bovine papilloma virus.

[0096] The transcription units encoding the corresponding DNA’s can also be introduced into animal cells together with another recombinant gene which may function as a dominant selectable marker in these cells in order to facilitate the isolation of specific cell clones which have integrated the recombinant DNA into their genome. Examples of this type of dominant selectable marker genes are Tn5 amino glycoside phosphotransferase, conferring resistance to geneticin (G418), hygromycin phosphotransferase, conferring resistance to hygromycin, and puromycin acetyl transferase, conferring resistance to puromycin. The recombinant expression vector encoding such a selectable marker can reside either on the same vector as the one encoding the cDNA of the desired protein, or it can be encoded on a separate vector which is simultaneously introduced and integrated to the genome of the host cell, frequently resulting in a tight physical linkage between the different transcription units.

[0097] Other types of selectable marker genes which can be used together with the cDNA of the desired protein are based on various transcription units encoding dihydrofolate reductase (dhfr). After introduction of this type of gene into cells lacking endogenous dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44), it will enable these to grow in media lacking nucleosides. An example of such a medium is Ham’s F12 without hypoxanthine, thymidin, and glycine. These dhfr-genes can be introduced together with the FVIII cDNA transcriptional units into CHO-cells of the above type, either linked on the same vector or on different vectors, thus creating dhfr-positive cell lines producing recombinant protein.

[0098] If the above cell lines are grown in the presence of the cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to methotrexate will emerge. These cell lines may produce recombinant protein at an increased rate due to the amplified number of linked dhfr and the desired protein’s transcriptional units. When propagating these cell lines in increasing concentrations of methotrexate (1-10000 nM), new cell lines can be obtained which produce the desired protein at very high rate.

[0099] The above cell lines producing the desired protein can be grown on a large scale, either in suspension culture or on various solid supports. Examples of these supports are micro carriers based on dextran or collagen matrices, or solid supports in the form of hollow fibres or various ceramic materials. When grown in cell suspension culture or on micro carriers the culture of the above cell lines can be performed either as a bath culture or as a perfusion culture with continuous production of conditioned medium over extended periods of time. Thus, according to the present invention, the above cell lines are well suited for the development of an industrial process for the production of the desired recombinant mutant proteins

Purification and Formulation [0100] The recombinant modified VWF protein, which accumulates in the medium of secreting cells of the above types, can be concentrated and purified by a variety of biochemical and chromatographic methods, including methods utilizing differences in size, charge, hydrophobicity, solubility, specific affinity, etc. between the desired protein and other substances in the cell cultivation medium.

[0101] An example of such purification is the adsorption of the recombinant mutant protein to a monoclonal antibody, directed to e.g. a HLEP, preferably human albumin, or directed to the respective coagulation factor, which is immobilised on a solid support. After adsorption of the modified VWF to the support, washing and desorption, the protein can be further purified by a variety of chromatographic techniques based on the above properties. The order of the purification steps is chosen e.g. according to capacity and selectivity of the steps, stability of the support or other aspects. Preferred purification steps e.g. are but are not limited to ion exchange chromatography steps, immune affinity chromatography steps, affinity chromatography steps, hydrophobic interaction chromatography steps, dye chromatography steps, hydroxyapatite chromatography steps, multimodal chromatography steps, and size exclusion chromatography steps.

[0102] In order to minimize the theoretical risk of virus contaminations, additional steps may be included in the process that allow effective inactivation or elimination of viruses. Such steps e.g. are heat treatment in the liquid or solid state, treatment with solvents and/or detergents, radiation in the visible or UV spectrum, gamma-radiation or nanofiltration.

[0103] The modified polynucleotides (e.g. DNA) of this invention may also be integrated into a transfer vector for use in the human gene therapy.

[0104] The various embodiments described herein may be combined with each other. The present invention will be further described in more detail in the following examples thereof. This description of specific embodiments of the invention will be made in conjunction with the appended figures.

[0105] The modified VWF as described in this invention can be formulated into pharmaceutical preparations for therapeutic use. The purified protein may be dissolved in conventional physiologically compatible aqueous buffer solutions to which there may be added, optionally, pharmaceutical excipients to provide pharmaceutical preparations.

[0106] Such pharmaceutical carriers and excipients as well as suitable pharmaceutical formulations are well known in the art (see for example "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis (2000) or "Handbook of Pharmaceutical Excipients", 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). In particular, the pharmaceutical composition comprising the polypeptide variant of the invention may be formulated in lyophilized or stable liquid form. The polypeptide variant may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

[0107] Formulations of the composition are delivered to the individual by any pharmaceutically suitable means of administration. Various delivery systems are known and can be used to administer the composition by any convenient route. Preferentially, the compositions of the invention are administered systemically. For systemic use, insertion proteins of the invention are formulated for parenteral (e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonar, intranasal ortransdermal) or enteral (e.g., oral, vaginal or rectal) delivery according to conventional methods. The most preferential routes of administration are intravenous and subcutaneous administration. The formulations can be administered continuously by infusion or by bolus injection. Some formulations encompass slow release systems.

[0108] The insertion proteins of the present invention are administered to patients in a therapeutically effective dose, meaning a dose that is sufficient to produce the desired effects, preventing or lessening the severity or spread of the condition or indication being treated without reaching a dose which produces intolerable adverse side effects. The exact dose depends on many factors as e.g. the indication, formulation, mode of administration and has to be determined in preclinical and clinical trials for each respective indication.

[0109] The pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical. One example of such an agent is the combination of non-modified FVIII with modified VWF.

Figures [0110]

Figure 1: Antigen and activity levels of wild-type FVIII and FVIII-C-terminal albumin fusion polypeptides

Figure 2: Comparison of human FVI11: Ag pharmacokinetics in VWF ko mice following i.v. injection of 100 U (FVIII :Ag)/kg FVIII wildtype and FVIII-FP 1656 VWF (mean; n=4/ti me point)

Figure 3: VWF:RCoA/WF:Ag ratios of cell culture supernatants containing wt rVWF (1570/1212), rVWF-FP (1572/1212) containing C-terminally linked albumin, or a mixed expression cell culture containing a mixture of wt rVWF (1570/1212) and rVWF-FP (1572/1212) transfected in a ratio of 5:1. Values of about 0,8 were obtained in every case that are close to 1 which is the theoretical ratio of NHP according to the unit definitions.

Figure 4: SDS-Agarose gel electrophoresis of wild-type rVWF (1570/1212) expressed in HEK cells (B) and rVWF-FP (1572/1212) expressed also in HEK cells (A). Bands were detected using either antibodies to VWF or to albumin (HSA).

Figure 5: Comparison of human rWVF wildtype and rVWF-FP pharmacokinetics following i.v. injection of 100 IU VWF:Ag in rats (mean, n=2-3 /timepoint)

Examples:

Example 1: Generation of expression vectors for FVIII molecules with C-terminal albumin fusion (not part of the invention) [0111] An expression plasmid based on plRESpuro3 (BD Biosciences) containing the full length FVIII cDNA sequence in its multiple cloning site (pF8-FL) was first used to create a B domain deleted FVIII. For that oligonucleotides F8-1 and F8-2 (SEQ ID NO 1 and 2) were used in a site-directed mutagenesis experiment according to standard protocols (QuickChange XL Site Directed Mutagenesis Kit, Stratagene, La Jolla, CA, USA) using pF8-FL as a template to delete the B domain. In a second step a sequence encoding the amino acid sequence RRGR was introduced to connect R740 of the A2 domain with R1648 of the a3 domain. This was performed in another round of site-directed mutagenesis using primers F8-3 and F8-4 (SEQ ID NO 3 and 4). The resulting plasmid was called pF8-457. A FVIII albumin fusion construct was generated stepwise. First, a PinAI cleavage site was introduced at the FVIII 3’terminus. For that a PCR fragment was generated using pF8-457 as template, using PCR primers We2827 and We2828 (SEQ ID NO 5 and 6), which was subsquently gel-purified, cut by restriction endonucleases BspE1 and Notl and ligated into pF8-457 previously digested with BspE1 and Notl. The resulting plasmid (pF8-1433) was then cut with enzymes PinAI and Notl and a fragment obtained by PCR on a human albumin cDNA containing plasmid using primers We 2829 and We 2830 (SEQ ID NO 7 and 8) and subsequently digested with enzymes PinAI and Notl was inserted. The resulting expression plasmid (pF8-1434) contained the coding sequences for a B domain deleted FVIII followed by a PinAI site to insert linkers (encoding the amino acid sequence ThrGly) and the coding sequence for human albumin. The amino acid sequence encoded by pF8-1434 is depicted as SEQ ID NO 9.

[0112] Linker sequences separating the FVIII and albumin moieties could then easily be inserted into the newly created PinAI site described above. The insertion of two linker sequences is described in the following. In addition, based on pF8-1434, theTG linker might be deleted in completion and even deletions into the C-terminus of FVIII or the N-terminus of albumin can be performed using site directed mutagenesis.

Insertion of a cleavable linker, derived from the FVIII thrombin cleavage site: First a PCR fragment containing the sequence encoding the thrombin cleavage site at position 372 was generated by PCR using primers We2979 and We2980 (SEQ ID NO 10 and 11) and pF8-457 as template. This fragment was purified, digested with PinAI and ligated into PinAI digested pF8-1434. Sequencing verified insertion of correct orientation of the fragment, the resulting plasmid was called pF8-1563.

[0113] Insertion of a flexible glycine/serine linker: A PCR fragment containing the coding sequence for a 31 amino acid glycine/serine linker was amplified by PCR from pFVII-937 described in W02007/090584 using primers We2991 and We2992 (SEQ ID NO 12 and 13). This fragment was then purified, digested by restriction endonuclease PinAI and ligated into PinAI digested pF8-1434. Sequencing verified insertion of correct orientation of the fragment, the resulting plasmid was called pF8-1568.

[0114] Using the protocols and plasmids described above and by applying molecular biology techniques known to those skilled in the art (and as described e.g. in Current Protocols in Molecular Biology, Ausubel FM et al. (eds.) John Wiley & Sons, Inc.; http://www.currentprotocols.com/WileyCDA/) other constructs can be made by the artisan to replace albumin by another HLEP or insert any other linker into the described PinAI site. Transfer of the FVIII/albumin cDNA into suitable vectors like plRESneo3 (Invitrogen) and pEE12.4 (Lonza) permitted expression and selection of clones expressing the respective FVIII albumin fusion protein in CHO cells.

Example 2: Transfection and expression of FVIII and VWF proteins [0115] Expression plasmidsweregrownupin E.coliTOPIO (Invitrogen, Carlsbad, CA, USA) and purified using standard protocols (Qiagen, Hilden, Germany). HEK-293 (Invitrogen) cel Is were transfected using the Lipofectamine 2000 reagent (Invitrogen) and grown up in serum-free medium (Invitrogen 293 Express) in the presence of 4 μg/ml Puromycin and optionally 0.5 lU/ml VWF. CHO cells (CHO-S, Invitrogen; CHOK1SV, Lonza) were transfected using the Lipofectamine 2000 reagent (Invitrogen) and grown up in serum-free medium (Invitrogen CD CHO, 6 mM glutamine for CHO-S and CD-CHO for CHOK1SV) in the presence of 500-1000 μg/ml Geneticin (CHO-S only). For FVIII expression optionally 0.5 lU/ml VWF were added. For vWF expression an expression plasmid encoding PACE/furin (pFu-797) as described in WO2007/144173 was cotransfected. In another experiment two plasmids encoding VWF wild-type and VWF fused at the C-terminus to albumin were cotransfected with pFu-797 resulting in VWF multimeres with wild-type VWF monomers and albumin-fused VWF monomers (see figure 3). Transfected cell populations were spread through T-flasks into roller bottles or small scale fermenters from which supernatants were harvested for purification.

[0116] Table 2 lists HEK-293 expression data of the constructs described in example 1.

Table 2:

Example 3: Increased expression rate of FVIII albumin fusion protein (not part of the invention) [0117] Figure 1 summarizes the results of an expression study of a FVIII albumin fusion protein in serum-free cell culture. HEK-293 cells were transfected in triplicate with pF8-1434 (FVIII C-terminal albumin fusion) and pF8-457 (FVIII wild-type), respectively, seeded into T80 flasks with equal cell numbers and grown in the absence of stabilizing VWF. Culture supernatant was then harvested after 96, 120 and 144 hours and tested for FVIII activity.

[0118] The results demonstrated an expression enhancing effect of the albumin moiety when present as an integral part of the FVIII molecule in cell culture. Consequently, the productivity was clearly improved in the case of the fusion protein compared to wild-type FVIII (Figure 1).

Example 4: Purification of FVIII proteins (not part of the invention) [0119] To the expression supernatant containing the FVIII molecule a sufficient amount of an immune affinity resin was added to bind the FVIII activity almost completely. The immune affinity resin had been prepared by binding an appropriate anti-FVI II MAb covalently to Sephacryl S1000 resin used as a support. After washing of the resin it was filled into a chromatography column and washed again. Elution was done using a buffer containing 250 mM CaCI2 and 50% ethylene glycol.

[0120] The immune affinity chromatography (IAC) fractions containing FVIILC activity were pooled, dialyzed against formulation buffer (excipients: sodium chloride, sucrose, histidine, calcium chloride, and Tween 80), and concentrated. Samples were either stored frozen or freeze-dried using an appropriate freeze-drying cycle.

[0121] Alternatively, the FVIII containing cell culture supernatant is concentrated/purified by a first ion exchange chromatography followed by further purification using immune affinity chromatography (IAC). In this case the eluate of the ion exchange chromatography is loaded onto an IAC column using the above mentioned resin.

Example 5: Analysis of FVIII activity and antigen (not part of the invention) [0122] For activity determination of FVI ll:C in vitro either a clotting assay (e.g. Pathromtin SL reagent and FVIII deficient plasma delivered by Dade Behring, Germany) or a chromogenic assay (e.g. Coamatic FVIII:C assay delivered by Haemo-chrom) were used. The assays were performed according to the manufacturers instructions.

[0123] FVIII antigen (FVI 11 :Ag) was determined by an ELISA whose performance is known to those skilled in the art. Briefly, microplates were incubated with 100 μί per well of the capture antibody (sheep anti-human FVIII IgG, Cedarlane CL20035K-C, diluted 1:200 in Buffer A [Sigma C3041]) for 2 hours at ambient temperature. After washing plates three times with buffer B (Sigma P3563), serial dilutions of the test sample in sample diluent buffer (Cedarlane) as well as serial dilutions of a FVIII preparation (CSL Behring; 200 - 2 mU/mL) in sample diluent buffer (volumes per well: 100 μί) were incubated for two hours at ambient temperature. After three wash steps with buffer B, 100 μί of a 1:2 dilution in buffer B of the detection antibody (sheep anti-human FVIII IgG, Cedarlane CL20035K-D, peroxidase labelled) were added to each well and incubated for another hour at ambient temperature. After three wash steps with buffer B, 100 μί of substrate solution (1:10 (v/v) TMB OUVF : TMB Buffer OUVG, Dade Behring) were added perwell and incubated for 30 minutes at ambient temperature in the dark. Addition of 100 μί stop solution (Dade Behring, OSFA) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations oftest samples were then calculated using the standard curve with the FVIII preparation as reference.

Example 6: Assessment of Pharmacokinetics of FVIII-FP in VWF ko mice following a single i.v. injection (not part of the invention) [0124] In order to compare the pharmacokinetics of FVIII wildtype (DNA 457) and a C-terminal FVIII-FP (DNA 1656), both FVIII variants were administered intravenously to mice. A VWF ko mouse strain (Denis C. et al, Proc. Natl. Acad. Sci. USA, 1998, Vol 95, 9524-9529) was chosen because, amongst other functions, VWF serves as a carrier and stabilizing protein for FVIII, thereby protecting FVIII from premature degradation, e.g. by proteases, and from premature elimination from circulation. For unmodified FVIII an undisturbed interaction with VWF is essential as exemplified by hemophilia A cases, caused by mutation in the C terminal region resulting in decreasing binding to VWF. In the case of modified FVIII such binding may, however, be even unwanted, in orderto examine or achieve improved pharmacokinetics. Accordingly both products were injected i.v. at a dose of 100 U (FVIII:Ag)/kg as bolus to two groups of mice (Tab. 3). Blood was sampled retroorbitally at appropriate intervals starting at 5 minutes after application of the test substances and up to 24 hours. One blood sample/mouse was taken, processed to plasma and stored frozen at-20°C until analysis. Human FVIILAg concentration was quantified using an ELISA assay specific for human FVIII or by a mixed ELISA specific for human albumin and FVIII, respectively. The mean plasma concentration of the, for each timepoint pooled, samples was used for calculation of pharmacokinetic parameters. Half-live was calculated using the time points of the beta phase of elimination according to the formula t1/2 = In2 / k, whereas k is the slope of the regression line. The result is depicted in Figure 2. Surprisingly, FVIII-FP 1656 (t1/2 = 3,06 h, between 5 and 960 min) had an about 3-4 times longer terminal half-life as compared to FVIII wildtype (t1/2 = 0,8 h, between 5 and 240 min). In addition, the recovery of FVIII-FP 1656 was increased by about 20% as compared to wildtype FVIII (Tab. 4).

Table 3: Treatment groups for comparison of pharmacokinetics FVIII in VWF ko mice

Table 4: Bioavailability (%) of FVIII wildtype and modified FVIII, FVIII-FP 1656, upon i.v. injection into VWF ko mice

Example 7: Generation of expression vectors for VWF wild-type and VWF albumin fusion proteins [0125] An expression plasmid containing the full length VWF cDNA sequence in its multiple cloning site was generated first. For that the coding sequence of VWF was amplified by polymerase chain reaction (PCR) using primer set VWF+ and VWF- (SEQ ID NO. 17 and 18) under standard conditions known to those skilled in the art (and as described e.g. in Current Protocols in Molecular Biology, Ausubel FM et al. (eds.) John Wiley & Sons, Inc.; http://www.currentproto-cols.com/WileyCDA/) from a plasmid containing VWF cDNA (as obtainable commercially, e.g. pMT2-VWF from ATCC, No. 67122). The resulting PCR fragment was digested by restriction endonuclease EcoRI and ligated into expression vector plRESpuro3 (BD Biosciences, Franklin Lakes, NJ, USA) which had been linearized by EcoRI. The resulting expression plasmid containing the wild-type cDNA of VWF downstream of the CMV promoter was called pVWF-1570.

[0126] A PCR fragment containing the coding sequence for a 31 amino acid glycine/serine linker and the human albumin cDNA was amplified from pFVII-937 described in W02007/090584 using primers We2994 and We1335 (SEQ ID NO. 19 and 20). This PCR fragment was then digested by restriction endonuclease Notl and ligated into Notl digested pVWF-1570. The resulting plasmid containing the coding sequences of VWF wt, the linker sequence and human albumin was called pVWF-1574.

[0127] In order to achieve expression of a fusion protein several bases had to be deleted between VWF and the linker sequence. This was peformed by site directed mutagenesis according to standard protocols (QuickChange XL Site Directed Mutagenesis Kit, Stratagene, La Jolla, CA, USA) using oligonucleotides We2995 and We2996 (SEQ ID NO 21 and 22). The resulting expression plasmid called pVWF-1572 contained the coding sequences of VWF in frame with that of a 31 amino acid glycin/serine linker and human albumin. The amino acid sequence of the expressed rVWF-FP is outlined as SEQ ID No. 25. The amino acid sequence of the human VWF preproprotein is outlined as SEQ ID NO. 24.

[0128] Using the protocols and plasmids described above and by applying molecular biology techniques known to those skilled in the art (and as described e.g. in Current Protocols in Molecular Biology, ibid) other constructs can be made by the artisan for replacement of the albumin sequence by another FILEP sequence or the linker sequence by another linker sequence.

Example 8: Purification of VWF and VWF albumin fusion proteins [0129] Cell culture supernatants containing VWF wild-type (rVWF wt) or VWF albumin fusion protein (rVWF-FP) were sterile-filtered through a 0,2μηι filter and dialysed against equilibration buffer (EB; 10mM Tris-HCI, 10mM CaCI2, pH 7.0). This material was then applied to a Heparin Fractogel column equilibrated with EB. The column was washed with EB and VWF proteins were eluated with 500mM NaCI in EB. The elution peak was concentrated and dialysed against FB buffer (3g/L sodium chloride, 20 g/L glycine, 5.5 g/L trisodium citrate dihydrate, pH 7.0). Finally the material was sterile filtrated and frozen in aliquots. If needed, further purification steps were applied comprising anion and/or cation exchange chromatography, HIC and SEC.

Example 9: Analysis of VWF activity and antigen [0130] Samples were analysed by immunoturbidimetric determination of VWF:Ag (OPAB03, Siemens Healthcare Diagnostics, Marburg, Germany) and for collagen binding (Technozym VWF:CBA ELISA, Ref. 5450301 with calibrator set 5450310 and control set 5450312, Technoclone, Vienna, Austria) as described by the manufacturer.

[0131] VWF:RCo testing was done using the BC VWF reagent of Siemens Healthcare Diagnostics, Marburg, Germany according to the manufacturers description. The International Concentrate Standard was used as a primary standard preparation to calibrate an in-house standard preparation for day to day use.

[0132] The ratios of VWF:RCo and VWF:Ag assays are calculated in order to compare this parameter for different constructs tested. As is shown in figure 3 the VWF:RCoA/WF:Ag ratio was comparable for wt rVWF and the C-terminal rVWF-albumin fusion protein.

[0133] For pharmacokinetic analyses VWF antigen was determined by an ELISA whose performance is known to those skilled in the art. Briefly, microplates were incubated with 100 μί per well of the capture antibody (rabbit anti human VWF-lgG, Dako A0082 [Dako, Hamburg, Germany], diluted 1:2000 in bufFer A [Sigma C3041, Sigma-Aldrich, Munich, Germany]) overnight at ambient temperature. After washing plates three times with bufFer B (Sigma P3563), each well was incubated with 200 μί bufFer C (Sigma P3688) for 1.5 hours at ambient temperature (blocking). After another three wash steps with bufFer B, serial dilutions of the test sample in buffer B as well as serial dilutions of standard human plasma (ORKL21; 20 - 0.2 mU/mL; Siemens Healthcare Diagnostics, Marburg, Germany) in buffer B (volumes per well: 100 μί) were incubated for 1.5 hours at ambient temperature. After three wash steps with bufFer B, 100 μί of a 1:16000 dilution in buffer B of the detection antibody (rabbit anti human VWF-lgG, Dako P0226, peroxidase labelled) were added to each well and incubated for 1 hour at ambient temperature. After three wash steps with bufFer B, 100 μί of substrate solution (OUVF, Siemens Healthcare Diagnostics) were added per well and incubated for 30 minutes at ambient temperature in the dark. Addition of 100 μί undiluted stop dilution (OSFA, Siemens Healthcare Diagnostics) prepared the samples for reading in a suitable microplate reader at 450 nm wavelength. Concentrations of the test samples were then calculated using the standard curve with standard human plasma as reference.

Example 10: Multimer analysis of VWF and VWF albumin fusion proteins [0134] VWF Multimer analysis was performed by SDS-agarose gel electrophoresis as recently described (Tatewaki et al.,. Thromb. Res. 52: 23-32 (1988), and Metzner et al., Haemophilia 4 (Suppl. 3): 25-32 (1998)) with minor modifications. Briefly, after equilibration in running bufFer ready to use 1% agarose mini gels (BioRad) were used to standardize the method as far as possible. Comparable amounts of VWF antigen were subjected to electrophoresis on the SDS-agarose gels. After Western blotting the VWF protein bands were detected using anti-VWF (DAKO, prod. No. 0854) or anti-albumin antibodies followed by alkaline phosphatase labelled anti-IgG antibodies (SIGMA, prod. No. 1305) and colour reaction quantified by densitometry.

[0135] Using wild-type rVWF (1570/797) and rVWF-FP (1572/797) it could be demonstrated by Western blotting and detection using anti-albumin or anti VWF antibodies that rVWF-FP forms a regular multimer distribution detected both by anti-albumin and anti-VWF antibodies (Figure 4). This confirms that although every subunit of the multimeric VWF contains albumin, a regular VWF multimer pattern is formed. The albumin moiety obviously does neither inhibit the N-terminal dimerization nor the C-terminal multimerization of the VWF molecules.

Example 11: Assessment of pharmacokinetics of VWF and VWF albuminfusion protein in rats following a single i.v. injection [0136] rVWF-FP and rVWF wt were administered intravenously to a total of 4 CD rats each. The dose was 100 U (VWF:Ag)/kg body weight, at an injection volume of 4 mL/kg.

Blood samples were drawn retroorbitally at appropriate intervals starting at 5 minutes after application of the test substances, using an alternating sampling scheme, resulting in samples from 2 animals / timepoint (t=0, 5, 30, 90 min, 4h, 1 d for subset Nr. 1 and 0, 15 min, 1,2, 8 h and 2 d for subset Nr. 2). The scheme was designed to minimize potential effects of blood sampling on the plasma concentration to be quantified. Blood was processed to plasma and stored deep frozen until analysis. The VWF:Ag level in plasma was subsequently quantified by an ELISA as described in Example 9. The mean plasma concentration was used for calculation of pharmacokinetic parameters. Half-live was calculated using the time points of the beta phase of elimination according to the formula t1/2 = In2 / k, whereas k is the slope of the regression line.

[0137] The result is depicted in Figure 5 (n=2/timepoint; mean). The terminal half-lifes were calculated to be 32.4 min. for the rVWF-FP and 2.6 min. for rVWF wt. Recovery was also improved for the rVWF-FP with 42.1% compared to 16.1% for rVWF wt.

SEQUENCE LISTING

[0138]

<110> CSL BEHRING GMBH CSL BEHRING GMBH <120 Factor VIII with prolonged half-life <130 2008_M004_A145 <150 EP 08011429.1 <151 > 2008-06-24 <160> 25 <170> Patentln version 3.3 <210> 1 <211> 36 <212> DNA <213> Artificial <220> <223> Primer <400> 1 caatgccatt gaaccaagac gagaaataac tcgtac 36 <210> 2 <211> 36 <212> DNA <213> Artificial <220> <223> Primer <400> 2 gtacgagtta tttctcgtct tggttcaatg gcattg 36 <210> 3 <211> 48 <212> DNA <213> Artificial <220> <223> Primer <400> 3 caatgccatt gaaccaagac gtcgtggtcg acgagaaata actcgtac 48 <210> 4 <211> 48 <212> DNA <213> Artificial <220> <223> Primer <400> 4 gtacgagtta tttctcgtcg accacgacgt cttggttcaa tggcattg 48

<210> 5 <211> 23 <212> DNA <213> Artificial <220 <223> Primer <400 5 cattattccg gatcaatcaa tgc 23 <210> 6 <211> 38 <212> DNA <213> Artificial <220> <223> Primer <400> 6 acgcggccgc ggtaccggtg tagaggtcct gtgcctcg 38 <210> 7 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 7 gtgaccggtg atgcacacaa gagtgaggtt g 31 <210> 8 <211> 32 <212> DNA <213> Artificial <220> <223> Primer <400> 8 cacgcggccg cctataagcc taaggcagct tg 32 <210> 9 <211> 2016 <212> PRT <213> Homo sapiens <400> 9

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala val Glu Leu ser Trp Asp Tyr 1 5 10 15

Met Gin Ser Asp Leu Gly Glu Leu Pro val Asp Ala Arg Phe Pro Pro 20 25 30

Arg val Pro Lys ser Phe Pro Phe Asn Thr ser val Val Tyr Lys Lys 35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn ile Ala Lys Pro 50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr ile Gin Ala Glu Val 65 70 75 80

Tyr Asp Thr val val ile Thr Leu Lys Asn Met Ala Ser His Pro val 85 90 95

Ser Leu His Ala val Gly val Ser Tyr Trp Lys Ala Ser Glu Gly Ala 100 105 110

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys val 115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140

Gly pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160

His val Asp Leu val Lys Asp Leu Asn Ser Gly Leu ile Gly Ala Leu 165 170 175

Leu val cys Arg Glu Gly ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190

His Lys Phe ile Leu Leu Phe Ala val Phe Asp Glu Gly Lys ser Trp 195 200 205

His ser Glu Thr Lys Asn Ser Leu Met Gin Asp Arg Asp Ala Ala Ser 210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr val Asn Arg 225 230 235 240

Ser Leu Pro Gly Leu ile Gly Cys His Arg Lys ser val Tyr Trp His 245 250 255

Val ile Gly Met Gly Thr Thr Pro Glu val His Ser Ile Phe Leu Glu 260 265 270

Giv His Thr Phe Leu val Arg Asn His Arg Gin Ala Ser Leu Glu Ile 275 280 285

Ser Pro Ile Thr Phe Leu Thr Ala Gin Thr Leu Leu Met Asp Leu Gly 290 295 300

Gin Phe Leu Leu Phe cys His Ile Ser Ser His Gin His Asp Gly Met 305 310 315 320

Glu Ala Tyr val Lys Val Asp Ser Cys Pro Glu Glu Pro Gin Leu Arg 325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350

Ser Glu Met Asp val val Arg Phe Asp Asp Asp Asn Ser pro ser Phe 355 360 365

Ile Gin ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp val His 370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu val Leu 385 390 395 400

Ala Pro Asp Asp Arg ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro 405 410 415

Gin Arg ile Gly Arg Lys Tyr Lys Lys Val Arg phe Met Ala Tyr Thr 420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala ile Gin His Glu ser Gly ile 435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu val Gly Asp Thr Leu Leu ile ile 450 455 460

Phe Lys Asn Gin Ala ser Arg Pro Tyr Asn ile Tyr Pro His Gly Ile 465 470 475 480

Thr Asp val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lvs Giv Val Lys 485 490 495

His Leu Lys Asp Phe pro Ile Leu Pro Gly Glu ile Phe lvs Tvr Lys 500 505 510

Trp Thr val Thr val Glu Asp Gly Pro Thr Lys Ser asp Pro Ara Cys 515 520 525

Leu Thr Arg Tyr Tyr ser Ser Phe val Asn Met Glu Arg Asp Leu Ala

Ser Gly Leu Ile Gly Pro Leu Leu Ile cys Tyr Lys Glu ser Val Asd 545 550 555 560

Gin Arg Gly Asn Gin ile Met ser Asp Lys Arg Asn val ile Leu Phe 565 570 575

Ser val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn ile Gin 580 585 5g0

Arg Phe Leu Pro Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe 595 600 605

Gin Ala ser Asn ile Met His Ser ile Asn Gly Tyr val Phe Asp Ser 610 615 620

Leu Gin Leu Ser val cys Leu His Glu val Ala Tyr Trp Tyr lie Leu 625 630 635 640 ser lie Gly Ala Gin Thr Asp Phe Leu Ser val Phe Phe Ser Gly Tyr 645 650 655

Thr Phe Lys His Lys Met val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670

Phe Ser Gly Glu Thr val Phe Met ser Met Glu Asn Pro Gly Leu Trp 675 680 685 lie Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700

Leu Leu Lys val ser ser cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720

Asp Ser Tyr Glu Asp lie Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala 725 730 735 lie Glu Pro Arg Arg Arg Gly Arg Arg Glu lie Thr Arg Thr Thr Leu 740 745 750

Gin ser Asp Gin Glu Glu lie Asp Tyr Asp Asp Thr lie Ser val Glu 755 760 765

Met Lys Lys Glu Asp Phe Asp lie Tyr Asp Glu Asp Glu Asn Gin ser 770 775 780

Pro Arg Ser Phe Gin Lys Lys Thr Arg His Tyr Phe lie Ala Ala Val 785 790 795 800

Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser ser Pro His val Leu Arg 805 810 815

Asn Arg Ala Gin ser Gly Ser Val Pro Gin Phe Lys Lys Val Val Phe 820 825 830

Gin Glu Phe Thr Asp Gly Ser Phe Thr Gin Pro Leu Tyr Arg Gly Glu 835 840 845

Leu Asn Glu His Leu Gly Leu Leu Gly Pro Tyr lie Arg Ala Glu val 850 855 860

Glu Asp Asn lie Met val Thr Phe Arg Asn Gin Ala Ser Arg Pro Tyr 865 870 875 880

Ser Phe Tyr Ser Ser Leu lie Ser Tyr Glu Glu Asp Gin Arg Gin Gly 885 890 895

Ala Glu Pro Arg Lys Asn Phe val Lys Pro Asn Glu Thr Lys Thr Tyr 900 905 910

Phe Trp Lys val Gin His His Met Ala Pro Thr Lys Asp Glu Phe Asp 915 920 925

Cys Lys Ala Trp Ala Tyr Phe ser Asp val Asp Leu Glu Lys Asp val 930 935 940

His Ser Gly Leu Ile Gly Pro Leu Leu val Cys His Thr Asn Thr Leu 945 950 955 960

Asn Pro Ala His Gly Arg Gin val Thr Val Gin Glu Phe Ala Leu Phe 965 970 975

Phe Thr ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met 980 985 990

Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gin Met Glu Asp Pro Thr 995 1000 1005

Phe Lys Glu Asn Tyr Arg Phe His Ala ile Asn Gly Tyr ile Met 1010 1015 1020

Asp Thr Leu Pro Gly Leu val Met Ala Gin Asp Gin Arg Ile Arg 1025 1030 1035

Trp Tyr Leu Leu ser Met Gly Ser Asn Glu Asn Ile His Ser Ile 1040 1045 1050

His Phe Ser Gly His Val Phe Thr val Arg Lys Lys Glu Glu Tyr 1055 1060 1065

Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly val Phe Glu Thr val 1070 1075 1080

Glu Met Leu Pro ser Lys Ala Gly ile Trp Arg val Glu Cys Leu 1085 1090 1095

Ile Gly Glu His Leu His Ala Gly Met ser Thr Leu Phe Leu val lioo 1105 mo

Tyr Ser Asn Lys Cys Gin Thr Pro Leu Gly Met Ala ser Gly His 1115 1120 1125

Ile Arg Asp Phe Gin Ile Thr Ala Ser Gly Gin Tyr Gly Gin Trp 1130 1135 1140

Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser ile Asn Ala 1145 1150 1155

Trp ser Thr Lys Glu Pro Phe Ser Trp ile Lys val Asp Leu Leu 1160 1165 1170

Ala Pro Met Ile ile His Gly ile Lys Thr Gin Gly Ala Arg Gin 1175 1180 1185

Lys Phe Ser Ser Leu Tyr ile Ser Gin Phe Ile Ile Met Tyr ser 1190 1195 1200

Leu Asp Gly Lys Lys Trp Gin Thr Tyr Arg Gly Asn Ser Thr Gly 1205 1210 1215

Thr Leu Met val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys 1220 1225 1230

His Asn Ile Phe Asn Pro pro Ile Ile Ala Arg Tyr ile Arg Leu 1235 1240 1245

His Pro Thr His Tyr Ser ile Arg Ser Thr Leu Arg Met Glu Leu 1250 1255 1260

Met Gly cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu 1265 1270 1275

Ser Lys Ala Ile ser Asp Ala Gin Ile Thr Ala Ser Ser Tyr Phe 1280 1285 1290

Thr Asn Met Phe Ala Thr Trp Ser Pro ser Lys Ala Arg Leu His 1295 1300 1305

Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val Asn Asn Pro 1310 1315 1320

Lys Glu Trp Leu Gin Val Asp Phe Gin Lys Thr Met Lys val Thr 1325 1330 1335

Gly val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr Ser Met Tyr 1340 1345 1350 val Lys Glu Phe Leu ile ser Ser Ser Gin Asp Gly His Gin Trp 1355 1360 1365

Thr Leu Phe Phe Gin Asn Gly Lys val Lys val Phe Gin Gly Asn 1370 1375 1380

Gin Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu 1385 1390 1395

Leu Thr Arg Tyr Leu Arg Ile His Pro Gin Ser Trp Val His Gin 1400 1405 1410

Ile Ala Leu Arg Met Glu val Leu Gly Cys Giu Ala Gin Asp Leu 1415 1420 1425

Tyr Thr Gly Asp Ala His Lys ser Glu val Ala His Arg Phe Lys 1430 1435 1440

Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu ile Ala Phe 1445 1450 1455

Ala Gin Tyr Leu Gin Gin Cys Pro Phe Glu Asp His Val Lys Leu 1460 1465 1470

Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu 1475 1480 1485

Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 1490 1495 1500

Lys Leu Cys Thr val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met 1505 1510 1515

Ala Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn Glu Cys Phe 1520 1525 1530

Leu Gin His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu val Arg 1535 1540 1545

Pro Glu Val Asp val Met cys Thr Ala Phe His Asp Asn Glu Glu 1550 1555 1560

Thr Phe Leu Lys Lys Tyr Leu Tyr Glu ile Ala Arg Arg His Pro 1565 1570 1575

Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys 1580 1585 1590

Ala Ala Phe Thr Glu Cys cys Gin Ala Ala Asp Lys Ala Ala Cys 1595 1600 1605

Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala ser 1610 1615 1620

Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser Leu Gin Lys Phe Gly 1625 1630 1635

Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gin Arg 1640 1645 1650

Phe Pro Lys Ala Glu Phe Ala Glu val Ser Lys Leu val Thr Asp 1655 1660 1665

Leu Thr Lys val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu 1670 1675 1680

Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn 1685 1690 1695

Gin Asp ser ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 1700 1705 1710

Leu Leu Glu Lys Ser His Cys Ile Ala Glu val Glu Asn Asp Glu 1715 1720 1725

Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 1730 1735 1740

Lys Asp val cys Lys Asn Tyr Ala Glu Ala Lys Asp val Phe Leu 1745 1750 1755

Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser 1760 1765 1770 val val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu 1775 1780 1785

Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 1790 1795 1800

Val Phe Asp Glu Phe Lys Pro Leu val Glu Glu Pro Gin Asn Leu 1805 1810 1815

Ile Lys Gin Asn cys Glu Leu Phe Glu Gin Leu Gly Glu Tyr Lys 1820 1825 1830

Phe Gin Asn Ala Leu Leu val Arg Tyr Thr Lys Lys val Pro Gin 1835 1840 1845

Val Ser Thr Pro Thr Leu Val Glu val Ser Arg Asn Leu Gly Lys 1850 1855 1860 val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro 1865 1870 1875

Cvs Ala Glu Asp Tyr Leu Ser val val Leu Asn Gin Leu cys val 1880 1885 1890

Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 1895 1900 1905

Thr Glu Ser Leu val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 1910 1915 1920

Va^ G^U T^r T^r Va^ Pro Lys Glu phe Asn Ala Glu Thr Phe 1925 1930 1935

Thr Phe His Ala Asp ile Cys Thr Leu Ser Glu Lys Glu Arg Gin 1940 1945 1950

Ile Lys Lys Gin Thr Ala Leu val Glu Leu val Lys His Lys Pro 1955 1960 1965

Lys Ala Thr Lys Glu Gin Leu Lys Ala Val Met Asp Asp Phe Ala 1970 1975 1980

Ala Phe val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr cys 1985 1990 1995

Phe Ala Glu Glu Giy Lys Lys Leu val Ala Ala Ser Gin Ala Ala 2000 2005 2010

Leu Gly Leu 2015 <210> 10 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 10 o*i gcgaccggtg atgacaactc tccttccttt a <210> 11 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 11 0-1 gcgaccggtc caagttttag gatgcttctt g <210> 12 <211> 27 <212> DNA <213> Artificial <220> <223> Primer <400> 12 gcgaccggtt cgagcggggg atctggc 27 <210 13 <211> 28 <212> DNA <213> Artificial <220> <223> Primer <400> 13 gcgaccggtg gatcccgacc ctccagag 28 <210 14 <211> 7020 <212> DNA <213> Homo sapiens <400> 14 atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60 accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120 ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180 acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240 gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300 gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360 ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420 gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480 aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540 gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600 gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660 tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720 gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780 ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840 accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900 cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960 gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020 gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080 gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140 gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200 tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260 cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320 aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380 attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440 ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500 gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560 ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620 actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680 gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740 agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800 aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860 cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920 tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980 attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040 atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100 atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160 atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220 agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280 ttctcccaga attcaagaca ccctagcact aggcaaaagc aatttaatgc caccacaatt 2340 ccagaaaatg acatagagaa gactgaccct tggtttgcac acagaacacc tatgcctaaa 2400 atacaaaatg tctcctctag tgatttgttg atgctcttgc gacagagtcc tactccacat 2460 gggctatcct tatctgatct ccaagaagcc aaatatgaga ctttttctga tgatccatca 2520 cctggagcaa tagacagtaa taacagcctg tctgaaatga cacacttcag gccacagctc 2580 catcacagtg gggacatggt atttacccct gagtcaggcc tccaattaag attaaatgag 2640 aaactgggga caactgcagc aacagagttg aagaaacttg atttcaaagt ttctagtaca 2700 tcaaataatc tgatttcaac aattccatca gacaatttgg cagcaggtac tgataataca 2760 agttccttag gacccccaag tatgccagtt cattatgata gtcaattaga taccactcta 2820 tttggcaaaa agtcatctcc ccttactgag tctggtggac ctctgagctt gagtgaagaa 2880 aataatgatt caaagttgtt agaatcaggt ttaatgaata gccaagaaag ttcatgggga 2940 aaaaatgtat cgtcaacaga gagtggtagg ttatttaaag ggaaaagagc tcatggacct 3000 gctttgttga ctaaagataa tgccttattc aaagttagca tctctttgtt aaagacaaac 3060 aaaacttcca ataattcagc aactaataga aagactcaca ttgatggccc atcattatta 3120 attgagaata gtccatcagt ctggcaaaat atattagaaa gtgacactga gtttaaaaaa 3180 gtgacacctt tgattcatga cagaatgctt atggacaaaa atgctacagc tttgaggcta 3240 aatcatatgt caaataaaac tacttcatca aaaaacatgg aaatggtcca acagaaaaaa 3300 gagggcccca ttccaccaga tgcacaaaat ccagatatgt cgttctttaa gatgctattc 3360 ttgccagaat cagcaaggtg gatacaaagg actcatggaa agaactctct gaactctggg 3420 caaggcccca gtccaaagca attagtatcc ttaggaccag aaaaatctgt ggaaggtcag 3480 aatttcttgt ctgagaaaaa caaagtggta gtaggaaagg gtgaatttac aaaggacgta 3540 ggactcaaag agatggtttt tccaagcagc agaaacctat ttcttactaa cttggataat 3600 ttacatgaaa ataatacaca caatcaagaa aaaaaaattc aggaagaaat agaaaagaag 3660 gaaacattaa tccaagagaa tgtagttttg cctcagatac atacagtgac tggcactaag 3720 aatttcatga agaacctttt cttactgagc actaggcaaa atgtagaagg ttcatatgac 3780 ggggcatatg ctccagtact tcaagatttt aggtcattaa atgattcaac aaatagaaca 3840 aagaaacaca cagctcattt ctcaaaaaaa ggggaggaag aaaacttgga aggcttggga 3900 aatcaaacca agcaaattgt agagaaatat gcatgcacca caaggatatc tcctaataca 3960 agccagcaga attttgtcac gcaacgtagt aagagagctt tgaaacaatt cagactccca 4020 ctagaagaaa cagaacttga aaaaaggata attgtggatg acacctcaac ccagtggtcc 4080 aaaaacatga aacatttgac cccgagcacc ctcacacaga tagactacaa tgagaaggag 4140 aaaggggcca ttactcagtc tcccttatca gattgcctta cgaggagtca tagcatccct 4200 caagcaaata gatctccatt acccattgca aaggtatcat catttccatc tattagacct 4260 atatatctga ccagggtcct attccaagac aactcttctc atcttccagc agcatcttat 4320 agaaagaaag attctggggt ccaagaaagc agtcatttct tacaaggagc caaaaaaaat 4380 aacctttctt tagccattct aaccttggag atgactggtg atcaaagaga ggttggctcc 4440 ctggggacaa gtgccacaaa ttcagtcaca tacaagaaag ttgagaacac tgttctcccg 4500 aaaccagact tgcccaaaac atctggcaaa gttgaattgc ttccaaaagt tcacatttat 4560 cagaaggacc tattccctac ggaaactagc aatgggtctc ctggccatct ggatctcgtg 4620 gaagggagcc ttcttcaggg aacagaggga gcgattaagt ggaatgaagc aaacagacct 4680 ggaaaagttc cctttctgag agtagcaaca gaaagctctg caaagactcc ctccaagcta 4740 l' ttggatcctc ttgcttggga taaccactat ggtactcaga taccaaaaga agagtggaaa 4800 tcccaagaga agtcaccaga aaaaacagct tttaagaaaa aggataccat tttgtccctg 4860 aacgcttgtg aaagcaatca tgcaatagca gcaataaatg agggacaaaa taagcccgaa 4920 atagaagtca cctgggcaaa gcaaggtagg actgaaaggc tgtgctctca aaacccacca 4980 gtcttgaaac gccatcaacg ggaaataact cgtactactc ttcagtcaga tcaagaggaa 5040 attgactatg atgataccat atcagttgaa atgaagaagg aagattttga catttatgat 5100 gaggatgaaa atcagagccc ccgcagcttt caaaagaaaa cacgacacta ttttattgct 5160 gcagtggaga ggctctggga ttatgggatg agtagctccc cacatgttct aagaaacagg 5220 gctcagagtg gcagtgtccc tcagttcaag aaagttgttt tccaggaatt tactgatggc 5280 tcctttactc agcccttata ccgtggagaa ctaaatgaac atttgggact cctggggcca 5340 tatataagag cagaagttga agataatatc atggtaactt tcagaaatca ggcctctcgt 5400 ccctattcct tctattctag ccttatttct tatgaggaag atcagaggca aggagcagaa 5460 cctagaaaaa actttgtcaa gcctaatgaa accaaaactt acttttggaa agtgcaacat 5520 catatggcac ccactaaaga tgagtttgac tgcaaagcct gggcttattt ctctgatgtt 5580 gacctggaaa aagatgtgca ctcaggcctg attggacccc ttctggtctg ccacactaac 5640 acactgaacc ctgctcatgg gagacaagtg acagtacagg aatttgctct gtttttcacc 5700 atctttgatg agaccaaaag ctggtacttc actgaaaata tggaaagaaa ctgcagggct 5760 ccctgcaata tccagatgga agatcccact tttaaagaga attatcgctt ccatgcaatc 5820 aatggctaca taatggatac actacctggc ttagtaatgg ctcaggatca aaggattcga 5880 tggtatctgc tcagcatggg cagcaatgaa aacatccatt ctattcattt cagtggacat 5940 gtgttcactg tacgaaaaaa agaggagtat aaaatggcac tgtacaatct ctatccaggt 6000 gtttttgaga cagtggaaat gttaccatcc aaagctggaa tttggcgggt ggaatgcctt 6060 attggcgagc atctacatgc tgggatgagc acactttttc tggtgtacag caataagtgt 6120 cagactcccc tgggaatggc ttctggacac attagagatt ttcagattac agcttcagga 6180 caatatggac agtgggcccc aaagctggcc agacttcatt attccggatc aatcaatgcc 6240 tggagcacca aggagccctt ttcttggatc aaggtggatc tgttggcacc aatgattatt 6300 cacggcatca agacccaggg tgcccgtcag aagttctcca gcctctacat ctctcagttt 6360 atcatcatgt atagtcttga tgggaagaag tggcagactt atcgaggaaa ttccactgga 6420 accttaatgg tcttctttgg caatgtggat tcatctggga taaaacacaa tatttttaac 6480 cctccaatta ttgctcgata catccgtttg cacccaactc attatagcat tcgcagcact 6540 cttcgcatgg agttgatggg ctgtgattta aatagttgca gcatgccatt gggaatggag 6600 agtaaagcaa tatcagatgc acagattact gcttcatcct actttaccaa tatgtttgcc 6660 acctggtctc cttcaaaagc tcgacttcac ctccaaggga ggagtaatgc ctggagacct 6720 caggtgaata atccaaaaga gtggctgcaa gtggacttcc agaagacaat gaaagtcaca 6780 ggagtaacta ctcagggagt aaaatctctg cttaccagca tgtatgtgaa ggagttcctc 6840 atctccagca gtcaagatgg ccatcagtgg actctctttt ttcagaatgg caaagtaaag 6900 gtttttcagg gaaatcaaga ctccttcaca cctgtggtga actctctaga cccaccgtta 6960 ctgactcgct accttcgaat tcacccccag agttgggtgc accagattgc cctgaggatg 7020 <210> 15 <211 >2332

<212> PRT <213> Homo sapiens <400 15

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala val Glu Leu Ser Trp Asp Tyr 15 10 15

Met Gin ser Asp Leu Gly Glu Leu pro val Asp Ala Arg phe Pro Pro 20 25 30

Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys 35 40 45

Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile Ala Lys Pro 50 55 60

Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr ile Gin Ala Glu val 65 70 75 80

Tyr Asp Thr val val lie Thr Leu Lys Asn Met Ala ser His Pro Val 85 90 95

Ser Leu His Ala val Gly val Ser Tyr Trp Lys Ala ser Glu Gly Ala 100 105 110

Glu Tyr Asp Asp Gin Thr Ser Gin Arg Glu Lys Glu Asp Asp Lys Val 115 120 125

Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gin Val Leu Lys Glu Asn 130 135 140

Gly Pro Met Ala ser Asp Pro Leu cys Leu Thr Tyr ser Tyr Leu ser 145 150 155 160

His val Asp Leu val Lys Asp Leu Asn ser Gly Leu lie Gly Ala Leu 165 170 175

Leu val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gin Thr Leu 180 185 190

His Lys Phe lie Leu Leu Phe Ala val Phe Asp Glu Gly Lys Ser Trp 195 200 205

His ser Glu Thr Lys Asn Ser Leu Met Gin Asp Arg Asp Ala Ala Ser 210 215 220

Ala Arg Ala Trp Pro Lys Met His Thr val Asn Gly Tyr val Asn Arg 225 230 235 240

Ser Leu Pro Gly Leu lie Gly Cys His Arg Lys Ser val Tyr Trp His 245 250 255

Val lie Gly Met Gly Thr Thr Pro Glu val His Ser lie Phe Leu Glu 260 265 270

Gly His Thr Phe Leu Val Arg Asn His Arg Gin Ala Ser Leu Glu lie 275 280 285

Ser Pro lie Thr Phe Leu Thr Ala Gin Thr Leu Leu Met Asp Leu Gly 290 295 300

Gin Phe Leu Leu Phe Cys His lie Ser Ser His Gin His Asp Gly Met 305 310 315 320

Glu Ala Tyr val Lys Val Asp Ser cys Pro Glu Glu Pro Gin Leu Arg 325 330 335

Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp 340 345 350

Ser Glu Met Asp Val val Arg Phe Asp Asp Asp Asn ser Pro Ser Phe 355 360 365 ile Gin ile Arg ser val Ala Lys Lys His pro Lys Thr Trp val His 370 375 380

Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu val Leu 385 390 395 400

Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gin Tyr Leu Asn Asn Gly Pro 405 410 415

Gin Arg Ile Gly Arg Lys Tyr Lys Lys val Arg Phe Met Ala Tyr Thr 420 425 430

Asp Glu Thr Phe Lys Thr Arg Glu Ala ile Gin His Glu Ser Gly Ile 435 440 445

Leu Gly Pro Leu Leu Tyr Gly Glu val Gly Asp Thr Leu Leu Ile Ile 450 455 460

Phe Lys Asn Gin Ala Ser Arg pro Tyr Asn Ile Tyr Pro His Gly Ile 465 470 475 480

Thr Asp val Arg Pro Leu Tyr ser Arg Arg Leu Pro Lys Gly val Lys 485 490 495

His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu ile Phe Lys Tyr Lys 500 505 510

Trp Thr val Thr val Glu Asp Gly Pro Thr Lys ser Asp Pro Arg cys 515 520 525

Leu Thr Arg Tyr Tyr Ser ser Phe val Asn Met Glu Arg Asp Leu Ala 530 535 540

Ser Gly Leu ile Gly Pro Leu Leu ile cys Tyr Lys Glu ser val Asp 545 550 555 560

Gin Arg Gly Asn Gin ile Met ser Asp Lys Arg Asn val ile Leu Phe 565 570 575

Ser val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn ile Gin 580 585 590

Arg Phe Leu Pro Asn Pro Ala Gly Val Gin Leu Glu Asp Pro Glu Phe 595 600 605

Gin Ala Ser Asn ile Met His Ser ile Asn Gly Tyr val Phe Asp Ser 610 615 620

Leu Gin Leu Ser val cys Leu His Glu val Ala Tyr Trp Tyr Ile Leu 625 630 635 640 ser Ile Gly Ala Gin Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr 645 650 655

Thr Phe Lys His Lys Met val Tyr Glu Asp Thr Leu Thr Leu Phe Pro 660 665 670

Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp 675 680 685

Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala 690 695 700

Leu Leu Lys val Ser ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu 705 710 715 720

Asp Ser Tyr Glu Asp ile Ser Ala Tyr Leu Leu ser Lys Asn Asn Ala 725 730 735

Ile Glu Pro Arg Ser Phe Ser Gin Asn Ser Arg His Arg Ser Thr Arg 740 745 750

Gin Lys Gin Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys 755 760 765

Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gin Asn 770 775 780

Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gin Ser Pro Thr Pro 785 790 795 800

His Gly Leu Ser Leu ser Asp Leu Gin Glu Ala Lys Tyr Glu Thr Phe 805 810 815

Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp ser Asn Asn Ser Leu Ser 820 825 830

Glu Met Thr His Phe Arg Pro Gin Leu His His Ser Gly Asp Met Val 835 840 845

Phe Thr pro Glu Ser Gly Leu Gin Leu Arg Leu Asn Glu Lys Leu Gly 850 855 860

Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys val Ser ser 865 870 875 880

Thr ser Asn Asn Leu ile ser Thr ile Pro Ser Asp Asn Leu Ala Ala 885 890 895

Gly Thr Asp Asn Thr ser ser Leu Gly pro Pro ser Met pro val His 900 905 910

Tyr Asp Ser Gin Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro 915 920 925

Leu Thr Glu ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp 930 935 940

Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gin Glu Ser Ser Trp 945 950 955 960

Gly Lys Asn val ser Ser Thr Glu ser Gly Arg Leu Phe Lys Gly Lys 965 970 975

Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys 980 985 990 val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala 995 1000 1005

Thr Asn Arg Lys Thr His Ile Asp Gly Pro ser Leu Leu Ile Glu 1010 1015 1020

Asn Ser Pro ser Val Trp Gin Asn ile Leu Glu Ser Asp Thr Glu 1025 1030 1035

Phe Lys Lys val Thr Pro Leu ile His Asp Arg Met Leu Met Asp 1040 1045 1050

Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr 1055 1060 1065

Thr Ser Ser Lys Asn Met Glu Met Val Gin Gin Lys Lys Glu Gly 1070 1075 1080 pro ile Pro Pro Asp Ala Gin Asn Pro Asp Met Ser Phe Phe Lys 1085 1090 1095

Met Leu Phe Leu Pro Glu Ser Ala Arg Trp ile Gin Arg Thr His 1100 1105 1110

Gly Lys Asn Ser Leu Asn Ser Gly Gin Gly Pro Ser Pro Lys Gin 1115 1120 1125

Leu val ser Leu Gly pro Glu Lys ser val Glu Gly Gin Asn Phe 1130 1135 1140

Leu ser Glu Lys Asn Lys val val val Gly Lys Gly Glu Phe Thr 1145 1150 1155

Lys Asp val Gly Leu Lys Glu Met val Phe Pro Ser Ser Arg Asn 1160 1165 1170

Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu Asn Asn Thr His 1175 1180 1185

Asn Gin Glu Lys Lys Ile Gin Glu Glu Ile Glu Lys Lys Glu Thr 1190 1195 1200

Leu ile Gin Glu Asn val Val Leu Pro Gin Ile His Thr Val Thr 1205 1210 1215

Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230

Gin Asn val Glu Gly ser Tyr Asp Gly Ala Tyr Ala Pro val Leu 1235 1240 1245

Gin Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260

His Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu 1265 1270 1275

Gly Leu Gly Asn Gin Thr Lys Gin ile val Glu Lys Tyr Ala cys 1280 1285 1290

Thr Thr Arg Ile Ser Pro Asn Thr Ser Gin Gin Asn Phe Val Thr 1295 1300 1305

Gin Arg Ser Lys Arg Ala Leu Lys Gin Phe Arg Leu Pro Leu Glu 1310 1315 1320

Glu Thr Glu Leu Glu Lys Arg ile ile val Asp Asp Thr ser Thr 1325 1330 1335

Gin Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr 1340 1345 1350

Gin ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala ile Thr Gin Ser 1355 1360 1365

Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser ile Pro Gin Ala 1370 1375 1380

Asn Arg ser Pro Leu Pro Ile Ala Lys val Ser ser Phe Pro Ser 1385 1390 1395

Ile Arg Pro Ile Tyr Leu Thr Arg val Leu Phe Gin Asp Asn Ser 1400 1405 1410

Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly val 1415 1420 1425

Gin Glu ser Ser His Phe Leu Gin Gly Ala Lys Lys Asn Asn Leu 1430 1435 1440

Ser Leu Ala ile Leu Thr Leu Glu Met Thr Gly Asp Gin Arg Glu 1445 1450 1455 val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr Lys 1460 1465 1470

Lys val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr 1475 1480 1485 ser Gly Lys val Glu Leu Leu pro Lys val His ile Tyr Gin Lys 1490 1495 1500

Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu 1505 1510 1515

Asp Leu val Glu Gly Ser Leu Leu Gin Gly Thr Glu Gly Ala ile 1520 1525 1530

Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg 1535 1540 1545 val Ala Thr Glu Ser ser Ala Lys Thr Pro Ser Lys Leu Leu Asp 1550 1555 1560

Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gin ile Pro Lys Glu 1565 1570 1575

Glu Trp Lys Ser Gin Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys 1580 1585 1590

Lys Lys Asp Thr ile Leu ser Leu Asn Ala Cys Glu Ser Asn His 1595 1600 1605

Ala Ile Ala Ala ile Asn Glu Gly Gin Asn Lys Pro Glu Ile Glu 1610 1615 1620

Val Thr Trp Ala Lys Gin Gly Arg Thr Glu Arg Leu Cys Ser Gin 1625 1630 1635

Asn Pro Pro val Leu Lys Arg His Gin Arg Glu ile Thr Arg Thr 1640 1645 1650

Thr Leu Gin Ser Asp Gin Glu Glu ile Asp Tyr Asp Asp Thr ile 1655 1660 1665

Ser val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp 1670 1675 1680

Glu Asn Gin ser pro Arg ser phe Gin Lys Lys Thr Arg His Tyr 1685 1690 1695

Phe ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser Ser 1700 1705 1710

Ser Pro His Val Leu Arg Asn Arg Ala Gin Ser Gly Ser val Pro 1715 1720 1725

Gin Phe Lys Lys val val Phe Gin Glu Phe Thr Asp Gly ser Phe 1730 1735 1740

Thr Gin Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu 1745 1750 1755

Leu Gly Pro Tyr ile Arg Ala Glu Val Glu Asp Asn Ile Met Val 1760 1765 1770

Thr Phe Arg Asn Gin Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser 1775 1780 1785

Leu Ile Ser Tyr Glu Glu Asp Gin Arg Gin Gly Ala Glu Pro Arg 1790 1795 1800

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys 1805 1810 1815

Val Gin His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys 1820 1825 1830

Ala Trp Ala Tyr Phe Ser Asp val Asp Leu Glu Lys Asp val His 1835 1840 1845

Ser Gly Leu ile Gly Pro Leu Leu val Cys His Thr Asn Thr Leu 1850 1855 1860

Asn Pro Ala His Gly Arg Gin val Thr val Gin Glu Phe Ala Leu 1865 1870 1875

Phe Phe Thr ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu 1880 1885 1890

Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn Ile Gin Met Glu 1895 1900 1905

Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala ile Asn Gly 1910 1915 1920

Tyr ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gin Asp Gin 1925 1930 1935

Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile 1940 1945 1950

His Ser Ile His Phe Ser Gly His val Phe Thr val Arg Lys Lys 1955 1960 1965

Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe 1970 1975 1980

Glu Thr val Glu Met Leu Pro ser Lys Ala Gly ile Trp Arg val 1985 1990 1995

Glu Cys Leu ile Gly Glu His Leu His Ala Gly Met ser Thr Leu 2000 2005 2010

Phe Leu val Tyr Ser Asn Lys cys Gin Thr Pro Leu Gly Met Ala 2015 2020 2025

Ser Gly His ile Arg Asp Phe Gin Ile Thr Ala Ser Gly Gin Tyr 2030 2035 2040

Gly Gin Trp Ala Pro Lys Leu Ala Arg Leu His Tyr ser Gly ser 2045 2050 2055

Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe ser Trp ile Lys Val 2060 2065 2070

Asp Leu Leu Ala Pro Met ile ile His Gly ile Lys Thr Gin Gly 2075 2080 2085

Ala Arg Gin Lys Phe Ser Ser Leu Tyr ile Ser Gin Phe ile ile 2090 2095 2100

Met Tyr ser Leu Asp Gly Lys Lys Trp Gin Thr Tyr Arg Gly Asn 2105 2110 2115

Ser Thr Gly Thr Leu Met val Phe Phe Gly Asn Val Asp ser Ser 2120 2125 2130

Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr 2135 2140 2145

Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg 2150 2155 2160

Met Glu Leu Met Gly cys Asp Leu Asn Ser cys Ser Met Pro Leu 2165 2170 2175

Gly Met Glu Ser Lys Ala lie Ser Asp Ala Gin lie Thr Ala Ser 2180 2185 2190

Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala 2195 2200 2205

Arg Leu His Leu Gin Gly Arg Ser Asn Ala Trp Arg Pro Gin Val 2210 2215 2220

Asn Asn Pro Lys Glu Trp Leu Gin val Asp Phe Gin Lys Thr Met 2225 2230 2235

Lys Val Thr Gly val Thr Thr Gin Gly Val Lys Ser Leu Leu Thr 2240 2245 2250

Ser Met Tyr val Lys Glu Phe Leu lie ser Ser Ser Gin Asp Gly 2255 2260 2265

His Gin Trp Thr Leu Phe Phe Gin Asn Gly Lys Val Lys Val Phe 2270 2275 2280

Gin Gly Asn Gin Asp ser Phe Thr Pro Val val Asn Ser Leu Asp 2285 2290 2295

Pro Pro Leu Leu Thr Arg Tyr Leu Arg lie His Pro Gin ser Trp 2300 * 2305 2310

Val His Gin lie Ala Leu Arg Met Glu val Leu Gly cys Glu Ala 2315 2320 2325

Gin Asp Leu Tyr 2330 <210> 16 <211> 585

<212> PRT <213> Homo sapiens <400 16

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu lie Ala Phe Ala Gin Tyr Leu Gin 20 25 30

Gin Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu val Thr Glu 35 40 45

Phe Ala Lys Thr Cys val Ala Asp Glu ser Ala Glu Asn Cys Asp Lys 50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr val Ala Thr Leu 65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gin Glu Pro 85 90 95

Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu 100 105 no

Pro Arg Leu val Arg pro Glu val Asp val Met cys Thr Ala Phe His 115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu lie Ala Arg 130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp Lys Ala Ala 165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190

Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser Leu Gin Lys Phe Gly Glu 195 200 205

Arg Ala Phe Lys Ala Trp Ala val Ala Arg Leu ser Gin Arg Phe Pro 210 215 220

Lys Ala Glu Phe Ala Glu val ser Lys Leu val Thr Asp Leu Thr Lys 225 230 235 240

Val His Thr Glu Cys cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255

Arg Ala Asp Leu Ala Lys Tyr lie Cys Glu Asn Gin Asp Ser lie Ser 260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 cys lie Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro ser 290 295 300

Leu Ala Ala Asp Phe val Glu Ser Lys Asp val Cys Lys Asn Tyr Ala 305 H 310 315 320

Glu Ala Lys Asp val phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335

Arg His Pro Asp Tyr ser Val val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu val Glu Glu Pro 370 375 380

Gin Asn Leu ile Lys Gin Asn Cys Glu Leu Phe Glu Gin Leu Gly Glu 385 390 395 400

Tyr Lys Phe Gin Asn Ala Leu Leu val Arg Tyr Thr Lys Lys val Pro 405 410 415

Gin val Ser Thr Pro Thr Leu Val Glu val Ser Arg Asn Leu Gly Lys 420 425 430

Val Gly ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro cys 435 440 445

Ala Glu Asp Tyr Leu ser val val Leu Asn Gin Leu Cys val Leu His 450 455 460

Glu Lys Thr Pro val Ser Asp Arg val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480

Leu val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu val Asp Glu Thr 485 490 495

Tyr val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 ile Cys Thr Leu Ser Glu Lys Glu Arg Gin Ile Lys Lys Gin Thr Ala 515 520 525

Leu val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gin Leu 530 535 540

Lys Ala val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu val 565 570 575

Ala Ala Ser Gin Ala Ala Leu Gly Leu 580 585 <210> 17 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 17 ttcgaattcc cgcagccctc atttgcaggg 30 <210> 18 <211> 31 <212> DNA <213> Artificial <220> <223> Primer <400> 18 tccgaattcc ggcagcagca ggcacccatg c 31 <210> 19 <211> 25 <212> DNA <213> Artificial <220> <223> Primer <400> 19 gcggcggccg cgagccccat ttccc 25 <210> 20 <211 > 18 <212> DNA <213> Artificial <220> <223> Primer <400> 20 gagagggagt actcaccc 18 <210> 21 <211> 27 <212> DNA <213> Artificial <220> <223> Primer <400> 21 ggaagtgcag caagtcgagc gggggat 27 <210> 22 <211> 27 <212> DNA <213> Artificial <220 <223> Primer <400> 22 atcccccgct cgacttgctg cacttcc 27 <210> 23 <211> 585 <212> PRT <213> Homo sapiens <400> 23

Asp Ala His Lys ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 15 10 15

Glu Asn Phe Lys Ala Leu val Leu lie Ala Phe Ala Gin Tyr Leu Gin 20 25 30

Gin Cys Pro Phe Glu Asp His Val Lys Leu val Asn Glu Val Thr Glu 35 40 45

Phe Ala Lys Thr cys val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gin Glu Pro 85 90 95

Glu Arg Asn Glu Cys Phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu 100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met cys Thr Ala Phe His 115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu lie Ala Arg 130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gin Ala Ala Asp Lys Ala Ala 165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190

Ser Ala Lys Gin Arg Leu Lys Cys Ala Ser Leu Gin Lys Phe Gly Glu 195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gin Arg Phe Pro 210 215 220

Lys Ala Giu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu cys Ala Asp Asp 245 250 255

Arg Ala Asp Leu Ala Lys Tyr ile cys Glu Asn Gin Asp Ser Ile Ser 260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285

Cys Ile Ala Glu val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp val Cys Lys Asn Tyr Ala 305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335

Arg His Pro Asp Tyr Ser Val val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365

Cys Tyr Ala Lys val Phe Asp Glu Phe Lys Pro Leu val Glu Glu Pro 370 375 380

Gin Asn Leu Ile Lys Gin Asn cys Glu Leu Phe Glu Gin Leu Gly Glu 385 390 395 400

Tyr Lys Phe Gin Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys val Pro 405 410 415

Gin Val Ser Thr Pro Thr Leu val Glu val Ser Arg Asn Leu Gly Lys 420 425 430

Val Gly ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445

Ala Glu Asp Tyr Leu Ser val Val Leu Asn Gin Leu Cys Val Leu His 450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480

Leu val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu val Asp Glu Thr 485 490 495

Tyr val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 ile cys Thr Leu ser Glu Lys Glu Arg Gin ile Lys Lys Gin Thr Ala 515 520 525

Leu val Glu Leu val Lys His Lys Pro Lys Ala Thr Lys Glu Gin Leu 530 535 540

Lys Ala val Met Asp Asp Phe Ala Ala Phe val Glu Lys cys Cys Lys 545 550 555 560

Ala Asp Asp Lys Glu Thr cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575

Ala Ala Ser Gin Ala Ala Leu Gly Leu 580 585 <210> 24 <211> 2813

<212> PRT <213> homo sapiens <400 24

Met Ile Pro Ala Arg Phe Ala Gly val Leu Leu Ala Leu Ala Leu ile 1 5 10 15

Leu Pro Gly Thr Leu cys Ala Glu Gly Thr Arg Gly Arg Ser Ser Thr 20 25 30

Ala Arg Cys Ser Leu Phe Gly Ser Asp Phe val Asn Thr Phe Asp Gly 35 40 45

Ser Met Tyr Ser Phe Ala Gly Tyr Cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60

Cvs Gin Lys Arg ser Phe ser ile Ile Gly Asp Phe Gin Asn Gly Lys 65 70 75 80

Ara val Ser Leu ser val Tyr Leu Gly Glu Phe Phe Asp ile His Leu 3 85 90 95

Phe val Asn Gly Thr val Thr Gin Gly Asp Gin Arg Val Ser Met pro 100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125

Leu ser Giv Glu Ala Tyr Gly Phe val Ala Arg ile Asp Gly Ser Gly 130 135 140

Asn Phe Gin Val Leu Leu Ser Asp Arg Tyr Phe Asn Lys Thr Cys Gly 145 150 155 160

Leu Cys Gly Asn Phe Asn ile Phe Ala Glu Asp Asp Phe Met Thr Gin 165 170 175

Glu Gly Thr Leu Thr Ser Asp Pro Tyr Asp Phe Ala Asn ser Trp Ala 180 185 190

Leu Ser Ser Gly Glu Gin Trp cys Glu Arg Ala ser Pro Pro Ser Ser 195 200 205

Ser cys Asn Ile Ser ser Gly Glu Met Gin Lys Gly Leu Trp Glu Gin 210 215 220

Cys Gin Leu Leu Lys Ser Thr ser val Phe Ala Arg Cys His Pro Leu 225 230 235 240

Val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu Cys Glu 245 250 255

Cys Ala Gly Gly Leu Glu cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270

Arg Thr Cys Ala Gin Glu Gly Met val Leu Tyr Gly Trp Thr Asp His 275 280 285 ser Ala Cys Ser Pro val Cys Pro Ala Gly Met Glu Tyr Arg Gin Cys 290 295 300

Val ser Pro Cys Ala Arg Thr Cys Gin Ser Leu His ile Asn Glu Met 305 310 315 320

Cys Gin Glu Arg Cys val Asp Gly Cys Ser Cys Pro Glu Gly Gin Leu 325 330 335

Leu Asp Glu Gly Leu Cys val Glu Ser Thr Glu Cys Pro cys val His 340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr Ser Leu Ser Arg Asp Cys Asn 355 360 365

Thr Cys Ile Cys Arg Asn ser Gin Trp ile Cys ser Asn Glu Glu Cys 370 375 380

Pro Gly Glu Cys Leu val Thr Gly Gin ser His Phe Lys Ser Phe Asp 385 390 395 400

Asn Arg Tyr Phe Thr Phe ser Gly Ile Cys Gin Tyr Leu Leu Ala Arg 405 410 415

Asd Cys Gin Asp His Ser Phe ser ile val ile Glu Thr val Gin cys K 420 425 430

Ala asp Asp Arg Asp Ala val Cys Thr Arg ser val Thr val Arg Leu 435 440 445 pro Gly Leu His Asn Ser Leu val Lys Leu Lys His Gly Ala Gly val 450 455 460

Ala Met Asp Gly Gin Asp lie Gin Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480

Arg lie Gin His Thr val Thr Ala ser val Arg Leu ser Tyr Gly Glu 485 490 495

Asp Leu Gin Met Asp Trp Asp Gly Arg Gly Arg Leu Leu val Lys Leu 500 505 510

Ser Pro val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525

Gly Asn Gin Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540

Arg val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gin 545 550 555 560

Asp Leu Gin Lys Gin His ser Asp Pro cys Ala Leu Asn Pro Arg Met 565 570 575

Thr Arg Phe ser Glu Glu Ala Cys Ala Val Leu Thr Ser Pro Thr Phe 580 585 590

Glu Ala Cys His Arg Ala val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605

Arg Tyr Asp Val cys ser Cys Ser Asp Gly Arg Glu cys Leu Cys Gly 610 615 620

Ala Leu Ala Ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly val Arg val 625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gin 645 650 655 val Tyr Leu Gin Cys Glv Thr Pro cys Asn Leu Thr Cys Arg ser Leu 660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685

Cys Pro G^y Leu Tyr Met AsP G,u Ar9 Gly Asp Cys Val Pro Lys 690 695 700

Ala Gin Cys Pro Cys Tyr Tyr Asp Gly Glu Ile Phe Gin Pro Glu Asp 705 710 715 720 ile Phe Ser Asp His His Thr Met Cys Tyr cys Glu Asp Gly Phe Met 725 730 735

His Cys Thr Met ser Gly val pro Gly Ser Leu Leu Pro Asp Ala val 740 745 750

Leu ser Ser Pro Leu Ser His Arg Ser Lys Arg ser Leu ser Cys Arg 755 760 765

Pro Pro Met val Lys Leu Val Cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gin Asn Tyr Asp Leu Glu Cys Met 785 790 795 800

Ser Met Gly Cys val Ser Gly Cys Leu Cys Pro Pro Gly Met val Arg 805 810 815

His Glu Asn Arg Cys val Ala Leu Glu Arg Cys Pro Cys Phe His Gin 820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr val Lys ile Gly Cys Asn Thr 835 840 845 cys Val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His val Cys Asp 850 855 860

Ala Thr cys Ser Thr Ile Gly Met Ala His Tyr Leu Thr Phe Asp Gly 865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gin Tyr val Leu val Gin Asp 885 890 895

Tyr Cys Gly Ser Asn Pro Gly Thr Phe Arg ile Leu Val Gly Asn Lys 900 905 910

Gly Cys Ser His Pro ser Val Lys Cys Lys Lys Arg val Thr ile Leu 915 920 925 val Glu Gly Gly Glu ile Glu Leu Phe Asp Gly Glu val Asn val Lys 930 935 940

Arg pro Met Lys Asp Glu Thr His Phe Glu Val val Glu ser Gly Arg 945 950 955 960

Tvr ile ile Leu Leu Leu Gly Lys Ala Leu Ser val val Trp Asp Arg 965 970 975

His Leu Ser Ile ser Val Val Leu Lys Gin Thr Tyr Gin Glu Lys Val 980 985 990 cys Gly Leu Cys Gly Asn Phe Asp Gly ile Gin Asn Asn Asp Leu Thr 995 1000 1005 ser Ser Asn Leu Gin val Glu Glu Asp Pro val Asp Phe Gly Asn 1010 1015 1020

Ser Trp Lys Val Ser Ser Gin Cys Ala Asp Thr Arg Lys val Pro 1025 1030 1035

Leu Asp ser Ser Pro Ala Thr Cys His Asn Asn Ile Met Lys Gin 1040 1045 1050

Thr Met val Asp Ser Ser cys Arg Ile Leu Thr Ser Asp val Phe 1055 1060 1065

Gin Asp Cys Asn Lys Leu val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080

Cys ile Tyr Asp Thr Cys Ser Cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095 cys Phe Cys Asp Thr ile Ala Ala Tyr Ala His val cys Ala Gin 1100 1105 1110

His Gly Lys val val Thr Trp Arg Thr Ala Thr Leu cys Pro Gin 1115 1120 1125

Ser Cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu cys Glu 1130 1135 1140

Trp Arg Tyr Asn Ser Cys Ala Pro Ala Cys Gin Val Thr Cys Gin 1145 1150 1155

His Pro Glu Pro Leu Ala cys Pro Val Gin Cys Val Glu Gly Cys 1160 1165 1170

His Ala His Cys Pro Pro Gly Lys Ile Leu Asp Glu Leu Leu Gin 1175 1180 1185

Thr Cys val Asp Pro Glu Asp Cys Pro val Cys Glu val Ala Gly 1190 1195 1200

Arg Arg Phe Ala ser Gly Lys Lys val Thr Leu Asn Pro Ser Asp 1205 1210 1215

Pro Glu His Cys Gin Ile Cys His Cys Asp val val Asn Leu Thr 1220 1225 1230

Cys Glu Ala Cys Gin Glu Pro Gly Gly Leu val val Pro Pro Thr 1235 1240 1245

Asp Ala Pro Val Ser Pro Thr Thr Leu Tyr val Glu Asp Ile Ser 1250 1255 1260

Glu Pro Pro Leu His Asp Phe Tyr Cys ser Arg Leu Leu Asp Leu 1265 1270 1275

Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe 1280 1285 1290

Glu val Leu Lys Ala Phe val val Asp Met Met Glu Arg Leu Arg 1295 1300 1305

Ile Ser Gin Lys Trp Val Arg Val Ala Val Val Glu Tyr His Asp 1310 1315 1320

Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser 1325 1330 1335

Glu Leu Arg Arg Ile Ala Ser Gin Val Lys Tyr Ala Gly ser Gin 1340 1345 1350 val Ala ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gin Ile 1355 1360 1365

Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg ile Thr Leu Leu 1370 1375 1380

Leu Met Ala ser Gin Glu Pro Gin Arg Met ser Arg Asn Phe val 1385 1390 1395

Arg Tyr val Gin Gly Leu Lys Lys Lys Lys Val Ile val ile Pro 1400 1405 1410

Val Gly Ile Gly Pro His Ala Asn Leu Lys Gin Ile Arg Leu Ile 1415 1420 1425

Glu Lys Gin Ala Pro Glu Asn Lys Ala Phe val Leu ser ser val 1430 1435 1440

Asp Glu Leu Glu Gin Gin Arg Asp Glu Ile val ser Tyr Leu Cys 1445 1450 1455

Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met 1460 1465 1470

Ala Gin val Thr Val Gly Pro Gly Leu Leu Gly val Ser Thr Leu A 1475 1480 1485

Gly Pro Lys Arg Asn Ser Met val Leu Asp val Ala Phe Val Leu 1490 1495 1500

Glu Gly Ser Asp Lys ile Gly Glu Ala Asp Phe Asn Arg ser Lys 1505 1510 1515

Glu Phe Met Glu Glu val lie Gin Arg Met Asp val Gly Gin Asp 1520 1525 1530 ser lie His val Thr Val Leu Gin Tyr ser Tyr Met val Thr val 1535 1540 1545

Glu Tyr Pro Phe ser Glu Ala Gin Ser Lys Gly Asp lie Leu Gin 1550 1555 1560

Ara Val Arg Glu lie Arg Tyr Gin Gly Gly Asn Arg Thr Asn Thr 9 1565 1570 1575

Gly Leu Ala Leu Arg Tyr Leu Ser Asp His Ser Phe Leu val ser y 1580 1585 1590

Gin Gly Asp Arg Glu Gin Ala Pro Asn Leu val Tyr Met val Thr 1595 1600 1605

Gly Asn Pro Ala ser Asp Glu lie Lys Arg Leu Pro Gly Asp lie 1610 1615 1620

Gin Val val Pro lie Gly val Gly Pro Asn Ala Asn val Gin Glu 1625 1630 1635

Leu Glu Arg lie Gly Trp Pro Asn Ala Pro lie Leu lie Gin Asp 1640 1645 1650

Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu Val Leu Gin Arg 1655 1660 1665

Cys Cys Ser Gly Glu Gly Leu Gin He Pro Thr Leu Ser Pro Ala 1670 1675 1680 pro Asp cys ser Gin Pro Leu Asp val lie Leu Leu Leu Asp Gly 1685 1690 1695

Ser ser Ser Phe Pro Ala ser Tyr Phe Asp Glu Met Lys Ser Phe 1700 1705 1710

Ala Lys Ala Phe He ser Lys Ala Asn lie Gly Pro Arg Leu Thr 1715 1720 1725

Gin Val Ser val Leu Gin Tyr Gly Ser Ile Thr Thr ile Asp val 1730 1735 1740 pro Trp Asn val val Pro Glu Lys Ala His Leu Leu Ser Leu Val 1745 1750 1755

Asp val Met Gin Arg Glu Gly Gly Pro ser Gin ile Gly Asp Ala 1760 1765 1770

Leu Gly Phe Ala val Arg Tyr Leu Thr ser Glu Met His Gly Ala 1775 1780 1785

Arg Pro Gly Ala Ser Lys Ala val Val Ile Leu val Thr Asp val 1790 1795 1800 ser val Asp Ser val Asp Ala Ala Ala Asp Ala Ala Arg ser Asn 1805 1810 1815

Arg val Thr Val Phe Pro ile Gly ile Gly Asp Arg Tyr Asp Ala 1820 1825 1830

Ala Gin Leu Arg ile Leu Ala Gly Pro Ala Gly Asp ser Asn val 1835 1840 1845 val Lys Leu Gin Arg Ile Glu Asp Leu Pro Thr Met Val Thr Leu 1850 1855 I860

Gly Asn ser Phe Leu His Lys Leu cys ser Gly Phe val Arg ile 1865 1870 1875 cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp val Trp 1880 1885 1890

Thr Leu Pro Asp Gin cys His Thr val Thr Cys Gin Pro Asp Gly 1895 1900 1905

Gin Thr Leu Leu Lys Ser His Arg val Asn cys Asp Arg Gly Leu 1910 1915 1920

Arg Pro Ser Cys Pro Asn Ser Gin Ser Pro val Lys val Glu Glu 1925 1930 1935

Thr Cys Gly Cys Arg Trp Thr Cys Pro cys val Cys Thr Gly Ser 1940 1945 1950

Ser Thr Arg His ile Val Thr Phe Asp Gly Gin Asn Phe Lys Leu 1955 1960 1965

Thr Gly ser cys ser Tyr val Leu Phe Gin Asn Lys Glu Gin Asp 1970 1975 1980

Leu Glu Val He Leu His Asn Gly Ala Cys Ser Pro Gly Ala Arg 1985 1990 1995

Gin Gly Cys Met Lys Ser ile Glu val Lys His ser Ala Leu Ser 2000 2005 2010

Val Glu Leu His Ser Asp Met Glu val Thr val Asn Gly Arg Leu 2015 2020 2025

Val Ser Val Pro Tyr Val Gly Gly Asn Met Glu val Asn val Tyr 2030 2035 2040

Gly Ala Ile Met His Glu Val Arg Phe Asn His Leu Gly His Ile 2045 2050 2055

Phe Thr Phe Thr Pro Gin Asn Asn Glu Phe Gin Leu Gin Leu Ser 2060 2065 2070

Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly ile Cys 2075 2080 2085

Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly Thr val 2090 2095 2100

Thr Thr Asp Trp Lys Thr Leu val Gin Glu Trp Thr Val Gin Arg 2105 2110 2115

Pro Gly Gin Thr Cys Gin Pro ile Leu Glu Glu Gin cys Leu val 2120 2125 2130

Pro Asp Ser Ser His Cys Gin val Leu Leu Leu Pro Leu Phe Ala 2135 2140 2145

Glu Cys His Lys val Leu Ala Pro Ala Thr Phe Tyr Ala ile Cys 2150 2155 2160

Gin Gin Asp Ser Cys His Gin Glu Gin val cys Glu val ile Ala 2165 2170 2175

Ser Tyr Ala His Leu Cys Arg Thr Asn Gly val Cys Val Asp Trp 2180 2185 2190

Arg Thr Pro Asp Phe Cys Ala Met ser cys Pro Pro Ser Leu val 2195 2200 2205

Tyr Asn His cys Glu His Gly cys Pro Arg His Cys Asp Gly Asn 2210 2215 2220

Val ser ser cys Gly Asp His Pro Ser Glu Gly Cys Phe Cys Pro 2225 2230 2235

Pro Asp Lys val Met Leu Glu Gly ser cys Val Pro Glu Glu Ala 2240 2245 2250 cys Thr Gin Cys lie Gly Glu Asp Gly val Gin His Gin Phe Leu 2255 2260 2265

Glu Ala Trp val Pro Asp His Gin Pro Cys Gin lie Cys Thr cys 2270 2275 2280

Leu Ser Gly Arg Lys val Asn Cys Thr Thr Gin Pro cys Pro Thr 2285 2290 2295

Ala Lys Ala Pro Thr Cys Gly Leu Cys Glu Val Ala Arg Leu Arg 2300 2305 2310

Gin Asn Ala Asp Gin Cys Cys Pro Glu Tyr Glu Cys Val cys Asp 2315 2320 2325 pro val Ser Cys Asp Leu Pro Pro Val Pro His Cys Glu Arg Gly 2330 2335 2340

Leu Gin Pro Thr Leu Thr Asn pro Gly Glu Cys Arg Pro Asn Phe 2345 2350 2355

Thr cys Ala Cys Arg Lys Glu Glu Cys Lys Arg Val ser Pro Pro 2360 2365 2370

Ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gin Cys 2375 2380 2385

Cys Asp Glu Tyr Glu Cys Ala Cys Asn Cys val Asn Ser Thr Val 2390 2395 2400

Ser cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn Asp cys 2405 2410 2415

Gly Cys Thr Thr Thr Thr Cys Leu Pro Asp Lys Val cys Val His 2420 2425 2430

Arg ser Thr lie Tyr Pro Val Gly Gin Phe Trp Glu Glu Gly Cys 2435 2440 2445

Asp val Cys Thr Cys Thr Asp Met Glu Asp Ala val Met Gly Leu 2450 2455 2460

Arg Val Ala Gin Cys ser Gin Lys Pro Cys Glu Asp ser Cys Arg 2465 2470 2475

Ser Gly phe Thr Tyr val Leu His Glu Gly Glu cys Cys Gly Arg 2480 2485 2490

Cys Leu Pro Ser Ala Cys Glu Val Val Thr Gly Ser Pro Arg Gly 2495 2500 2505

Asp Ser Gin ser ser Trp Lys Ser val Gly ser Gin Trp Ala Ser 2510 2515 2520

Pro Glu Asn Pro cys Leu ile Asn Glu Cys val Arg val Lys Glu 2525 2530 2535

Glu val Phe lie Gin Gin Arg Asn val ser cys Pro Gin Leu Glu 2540 2545 2550

Val Pro val Cys Pro Ser Gly Phe Gin Leu Ser Cys Lys Thr ser 2555 2560 2565

Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala Cys Met 2570 2575 2580

Leu Asn Gly Thr Val lie Gly Pro Gly Lys Thr Val Met lie Asp 2585 2590 2595 val Cys Thr Thr cys Arg cys Met Val Gin val Gly Val lie Ser 2600 2605 2610

Gly Phe Lys Leu Glu cys Arg Lys Thr Thr Cys Asn Pro Cys Pro 2615 2620 2625

Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu cys Cys Gly Arg 2630 2635 2640

Cys Leu Pro Thr Ala Cys Thr lie Gin Leu Arg Gly Gly Gin lie 2645 2650 2655

Met Thr Leu Lys Arg Asp Glu Thr Leu Gin Asp Gly Cys Asp Thr 2660 2665 2670

His Phe Cys Lys Val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys 2675 2680 2685

Arq val Thr Gly Cys Pro Pro Phe Asp Glu His Lys Cys Leu Ala 2690 2695 2700

Glu Gly Gly Lys lie Met Lys lie Pro Gly Thr cys Cys Asp Thr 2705 2710 271¾

Cys Glu Glu Pro Glu Cys Asn Asp lie Thr Ala Arg Leu Gin Tyr 2720 2725 2730 val Lys val Gly ser cys Lys ser Glu val Glu val Asp lie His 2735 2740 2745

Tyr cys Gin Gly Lys cys Ala Ser Lys Ala Met Tyr Ser ile Asp 2750 2755 2760

Ile Asn Asp val Gin Asp Gin Cys ser Cys Cys Ser Pro Thr Arg 2765 2770 2775

Thr Glu Pro Met Gin val Ala Leu His Cys Thr Asn Gly ser val 2780 2785 2790 val Tyr His Glu val Leu Asn Ala Met Glu Cys Lys cys ser pro 2795 2800 2805

Arg Lys Cys Ser Lys 2810 <210> 25 <211> 3429 <212> PRT <213> Artificial <220 <223> Amino acid sequence of human VWF albumin fusion preproprotein <400 25

Met ile Pro Ala Arg Phe Ala Gly val Leu Leu Ala Leu Ala Leu lie J 1 lie 5 s 10 15

Leu Pro Gly Thr Leu Cys Ala Glu Gly Thr Arg Gly Arg Ser ser Thr 20 25 30

Ala Ara cys Ser Leu Phe Gly Ser Asp Phe Val Asn Thr Phe Asp Gly M 35 40 45 ser Met Tyr ser Phe Ala Gly Tyr cys Ser Tyr Leu Leu Ala Gly Gly 50 55 60

Cvs Gin lvs Arg ser Phe Ser ile ile Gly Asp Phe Gin Asn Gly Lys 65 70 75 80

Arq val Ser Leu Ser Val Tyr Leu Gly Glu Phe Phe Asp lie His Leu 85 90 95

Phe val Asn Gly Thr val Thr Gin Gly Asp Gin Arg val Ser Met Pro 100 105 110

Tyr Ala Ser Lys Gly Leu Tyr Leu Glu Thr Glu Ala Gly Tyr Tyr Lys 115 120 125

Leu Ser Gly Glu Ala Tyr Gly phe val Ala Arg lie Asp Gly ser Gly 130 135 140

Asn Phe Gin val Leu Leu Ser asp Arg Tyr Phe Asn Lys Thr cys Gly 145 150 μ 155 160

Leu Cys Gly Asn Phe Asn lie Phe Ala Glu Asp Asp Phe Met Thr Gin 165 170 175

Glu Gly Thr Leu Thr ser Asp Pro Tyr Asp Phe Ala Asn Ser Trp Ala 180 185 190

Leu Ser ser Gly Glu Gin Trp Cys Glu Arg Ala Ser pro Pro Ser Ser 195 200 205

Ser Cys Asn lie Ser ser Gly Glu Met Gin Lys Gly Leu Trp Glu Gin 210 215 220 cys Gin Leu Leu Lys ser Thr ser val Phe Ala Arg cys His Pro Leu 225 230 235 240 val Asp Pro Glu Pro Phe Val Ala Leu Cys Glu Lys Thr Leu cys Glu 245 250 255

Cys Ala Gly Gly Leu Glu Cys Ala Cys Pro Ala Leu Leu Glu Tyr Ala 260 265 270

Arg Thr cys Ala Gin Glu Gly Met val Leu Tyr Gly Trp Thr Asp His 275 280 285 ser Ala cys ser Pro val cys Pro Ala Gly Met Glu Tyr Arg Gin Cys 290 295 300

Val Ser Pro Cys Ala Arg Thr Cys Gin ser Leu His lie Asn Glu Met 305 310 315 320

Cys Gin Glu Arg Cys val Asp Gly cys ser Cys Pro Glu Gly Gin Leu 325 330 335

Leu Asp Glu Gly Leu Cys val Glu Ser Thr Glu Cys Pro Cys val His 340 345 350

Ser Gly Lys Arg Tyr Pro Pro Gly Thr ser Leu ser Arg Asp Cys Asn 355 360 365

Thr Cys lie Cys Arg Asn Ser Gin Trp lie Cys Ser Asn Glu Glu Cys 370 375 380

Pro Gly Glu cys Leu val Thr Gly Gin ser His Phe Lys Ser phe Asp 385 390 395 400

Asn Arg Tyr phe Thr Phe ser Gly il.e cys Gin Tyr Leu Leu Ala Arg 405 410 415

Asp Cys Gin Asp His ser Phe ser lie val lie Glu Thr val Gin cys 420 425 430

Ala Asp Asp Arg Asp Ala val Cys Thr Arg Ser val Thr val Arg Leu 435 440 445

Pro Gly Leu His Asn Ser Leu Val Lys Leu Lys His Gly Ala Gly val 450 455 460

Ala Met Asp Gly Gin Asp lie Gin Leu Pro Leu Leu Lys Gly Asp Leu 465 470 475 480

Arg lie Gin His Thr val Thr Ala Ser Val Arg Leu Ser Tyr Gly Glu 485 490 495

Asp Leu Gin Met Asp Trp Asp Gly Arg Gly Arg Leu Leu val Lys Leu 500 505 510

Ser Pro Val Tyr Ala Gly Lys Thr Cys Gly Leu Cys Gly Asn Tyr Asn 515 520 525

Gly Asn Gin Gly Asp Asp Phe Leu Thr Pro Ser Gly Leu Ala Glu Pro 530 535 540

Arg val Glu Asp Phe Gly Asn Ala Trp Lys Leu His Gly Asp Cys Gin 545 550 555 560

Asp Leu Gin Lys Gin His ser Asp Pro Cys Ala Leu Asn Pro Arg Met 565 570 575

Thr Arg Phe Ser Glu Glu Ala Cys Ala val Leu Thr Ser Pro Thr Phe 580 585 590

Glu Ala Cys His Arg Ala Val Ser Pro Leu Pro Tyr Leu Arg Asn Cys 595 600 605

Arg Tyr Asp val Cys Ser cys Ser Asp Gly Arg Glu cys Leu Cys Gly 610 615 620

Ala Leu Ala ser Tyr Ala Ala Ala Cys Ala Gly Arg Gly val Arg val 625 630 635 640

Ala Trp Arg Glu Pro Gly Arg Cys Glu Leu Asn Cys Pro Lys Gly Gin 645 650 655 val Tyr Leu Gin Cys Gly Thr Pro cys Asn Leu Thr Cys Arg Ser Leu 660 665 670

Ser Tyr Pro Asp Glu Glu Cys Asn Glu Ala Cys Leu Glu Gly Cys Phe 675 680 685

Cys Pro Pro Gly Leu Tyr Met Asp Glu Arg Gly Asp cys val Pro Lys 690 695 700

Ala Gin Cys Pro Cys Tyr Tyr Asp Gly Glu lie Phe Gin Pro Glu Asp 705 710 715 720 ile Phe Ser Asp His His Thr Met Cys Tyr Cys Glu Asp Gly Phe Met 725 730 735

His Cys Thr Met Ser Gly val Pro Gly Ser Leu Leu Pro Asp Ala Val 740 745 750

Leu Ser Ser pro Leu Ser His Arg ser Lys Arg ser Leu Ser Cys Arg 755 760 765 pro Pro Met val Lys Leu val cys Pro Ala Asp Asn Leu Arg Ala Glu 770 775 780

Gly Leu Glu Cys Thr Lys Thr Cys Gin Asn Tyr Asp Leu Glu cys Met 785 790 795 800

Ser Met Gly Cys Val Ser Gly Cys Leu cys Pro Pro Gly Met val Arg 805 810 815

His Glu Asn Arg Cys Val Ala Leu Glu Arg Cys Pro Cys Phe His Gin 820 825 830

Gly Lys Glu Tyr Ala Pro Gly Glu Thr val Lys lie Gly Cys Asn Thr 835 840 845

Cys val Cys Arg Asp Arg Lys Trp Asn Cys Thr Asp His Val cys Asp 850 855 860

Ala Thr Cys Ser Thr lie Gly Met Ala His Tyr Leu Thr Phe Asp Gly 865 870 875 880

Leu Lys Tyr Leu Phe Pro Gly Glu Cys Gin Tyr Val Leu val Gin Asp 885 890 895

Tvr cys Gly Ser Asn Pro Gly Thr Phe Arg lie Leu val Gly Asn Lys 3 900 905 910

Gly Cys ser His Pro Ser val Lys cys Lys Lys Arg val Thr lie Leu y 3 915 920 925 val Glu Gly Gly Glu lie Glu Leu Phe Asp Gly Glu val Asn val Lys 930 935 940

Arg Pro Met Lys Asp Glu Thr His Phe Glu val val Glu ser Gly Arg 945 950 955 960

Tvr lie lie Leu Leu Leu Gly Lys Ala Leu Ser val val Trp Asp Arg 3 965 970 975

His Leu ser ile Ser val val Leu Lys Gin Thr Tyr Gin Glu Lys val 980 985 990

Cys Gly Leu cys Gly Asn Phe Asp Gly ile Gin Asn Asn Asp Leu Thr 995 1000 1005

Ser ser Asn Leu Gin val Glu Glu Asp Pro val Asp Phe Gly Asn 1010 1015 1020

Ser Trp Lys val Ser Ser Gin Cys Ala Asp Thr Arg Lys val Pro 1025 1030 1035

Leu Asp Ser ser pro Ala Thr Cys His Asn Asn ile Met Lys Gin 1040 1045 1050

Thr Met val Asp ser ser cys Arg Ile Leu Thr ser Asp Val Phe 1055 1060 1065

Gin Asp Cys Asn Lys Leu Val Asp Pro Glu Pro Tyr Leu Asp Val 1070 1075 1080

Cys Ile Tyr Asp Thr Cys ser cys Glu Ser Ile Gly Asp Cys Ala 1085 1090 1095

Cys Phe Cys Asp Thr Ile Ala Ala Tyr Ala His Val Cys Ala Gin 1100 1105 1110

His Gly Lys val Val Thr Trp Arg Thr Ala Thr Leu cys Pro Gin 1115 1120 1125

Ser cys Glu Glu Arg Asn Leu Arg Glu Asn Gly Tyr Glu Cys Glu 1130 1135 1140

Trp Arg Tyr Asn Ser cys Ala Pro Ala cys Gin val Thr Cys Gin 1145 1150 1155

His Pro Glu Pro Leu Ala Cys Pro val Gin Cys val Glu Gly Cys 1160 1165 1170

His Ala His Cys Pro Pro Gly Lys ile Leu Asp Glu Leu Leu Gin 1175 1180 1185

Thr Cys Val Asp Pro Glu Asp Cys Pro Val Cys Glu val Ala Gly 1190 1195 1200

Arg Arg Phe Ala ser Gly Lys Lys val Thr Leu Asn pro Ser Asp 1205 1210 1215

Pro Glu His Cys Gin Ile Cys His Cys Asp Val Val Asn Leu Thr 1220 1225 1230 cys Glu Ala Cys Gin Glu Pro Gly Gly Leu val val Pro Pro Thr 1235 1240 1245

Asp Ala Pro Val ser Pro Thr Thr Leu Tyr val Glu Asp lie Ser 1250 1255 1260

Glu Pro Pro Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu 1265 1270 1275

Val Phe Leu Leu Asp Gly Ser ser Arg Leu ser Glu Ala Glu Phe 1280 1285 1290

Glu val Leu Lys Ala Phe val val Asp Met Met Glu Arg Leu Arg 1295 1300 1305

Ile Ser Gin Lys Trp val Arg val Ala Val val Glu Tyr His Asp 1310 1315 1320

Gly Ser His Ala Tyr lie Gly Leu Lys Asp Arg Lys Arg Pro Ser 1325 1330 1335

Glu Leu Arq Arg lie Ala Ser Gin val Lys Tyr Ala Gly Ser Gin 1340 1345 1350 val Ala Ser Thr ser Glu val Leu Lys Tyr Thr Leu Phe Gin lie 1355 1360 1365

Phe Ser Lys lie Asp Arg Pro Glu Ala Ser Arg lie Thr Leu Leu 1370 1375 1380

Leu Met Ala ser Gin Glu Pro Gin Arg Met ser Arg Asn Phe Val 1385 1390 1395

Arg Tyr val Gin Gly Leu Lys Lys Lys Lys val lie val He Pro 1400 1405 1410

Val Gly lie Gly Pro His Ala Asn Leu Lys Gin lie Arg Leu lie 1415 1420 1425

Glu Lys Gin Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser val 1430 1435 1440

Asp Glu Leu Glu Gin Gin Arg Asp Glu He val ser Tyr Leu cys 1445 1450 1455

Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met 1460 1465 1470

Ala Gin val Thr val Gly Pro Gly Leu Leu Gly val ser Thr Leu 1475 1480 1485

Gly Pro Lys Arg Asn Ser Met val Leu Asp val Ala Phe val Leu 1490 1495 1500

Glu Gly Ser Asp Lys Ile Gly Glu Ala Asp Phe Asn Arg ser Lys 1505 1510 1515 *

Glu Phe Met Glu Glu val ile Gin Arg Met Asp val Gly Gin Asp 1520 1525 1530

Ser Ile His Val Thr Val Leu Gin Tyr ser Tyr Met Val Thr val 1535 1540 1545

Glu Tyr Pro Phe ser Glu Ala Gin Ser Lys Gly Asp Ile Leu Gin 1550 1555 1560

Arg Val Arg Glu ile Arg Tyr Gin Gly Gly Asn Arg Thr Asn Thr 1565 1570 1575

Gly Leu Ala Leu Arg Tyr Leu Ser Asp His ser Phe Leu val Ser 1580 1585 1590

Gin Gly Asp Arg Glu Gin Ala Pro Asn Leu val Tyr Met val Thr 1595 1600 1605

Gly Asn Pro Ala Ser Asp Glu Ile Lys Arg Leu Pro Gly Asp ile 1610 1615 1620

Gin val val Pro ile Gly val Gly Pro Asn Ala Asn val Gin Glu 1625 1630 1635

Leu Glu Arg ile Gly Trp Pro Asn Ala Pro Ile Leu ile Gin Asp 1640 1645 1650

Phe Glu Thr Leu Pro Arg Glu Ala Pro Asp Leu val Leu Gin Arg 1655 1660 1665

Cys cys Ser Gly Glu Gly Leu Gin ile Pro Thr Leu Ser Pro Ala 1670 1675 1680

Pro Asp Cys Ser Gin Pro Leu Asp Val Ile Leu Leu Leu Asp Gly 1685 1690 1695

Ser ser ser Phe Pro Ala Ser Tyr Phe Asp Glu Met Lys Ser Phe 1700 1705 1710

Ala Lys Ala Phe ile ser Lys Ala Asn ile Gly Pro Arg Leu Thr 1715 1720 1725

Gin val Ser val Leu Gin Tyr Gly Ser ile Thr Thr ile Asp val 1730 1735 1740

Pro Trp Asn val val Pro Glu Lys Ala His Leu Leu ser Leu val 1745 1750 1755

Asp val Met Gin Arg Glu Gly Gly Pro Ser Gin lie Gly Asp Ala 1760 1765 1770

Leu Gly Phe Ala val Arg Tyr Leu Thr Ser Glu Met His Gly Ala 1775 1780 1785

Arg Pro Gly Ala Ser Lys Ala val val lie Leu val Thr Asp Val 1790 1795 1800 ser val Asp Ser val Asp Ala Ala Ala Asp Ala Ala Arg ser Asn 1805 1810 1815

Arg val Thr Val Phe Pro lie Gly lie Gly Asp Arg Tyr Asp Ala 1820 1825 1830

Ala Gin Leu Arg lie Leu Ala Gly Pro Ala Gly Asp Ser Asn Val 1835 1840 1845 val Lys Leu Gin Arg lie Glu Asp Leu Pro Thr Met Val Thr Leu 1850 1855 1860

Gly Asn Ser Phe Leu His Lys Leu Cys ser Gly Phe Val Arg lie 1865 1870 1875 cys Met Asp Glu Asp Gly Asn Glu Lys Arg Pro Gly Asp val Trp 1880 1885 1890

Thr Leu Pro Asp Gin cys His Thr Val Thr Cys Gin Pro Asp Gly 1895 1900 1905

Gin Thr Leu Leu Lys Ser His Arg Val Asn Cys Asp Arg Gly Leu 1910 1915 1920

Arq Pro Ser Cys Pro Asn Ser Gin Ser Pro Val Lys val Glu Glu 1925 1930 1935

Thr cys Gly Cys Arg Trp Thr cys Pro Cys val Cys Thr Gly Ser 1940 1945 1950

Ser Thr Arg His lie val Thr Phe Asp Gly Gin Asn Phe Lys Leu 1955 I960 1965

Thr Gly Ser Cys Ser Tyr Val Leu Phe Gin Asn Lys Glu Gin Asp 1970 1975 1980

Leu Glu Val lie Leu His Asn Gly Ala Cys Ser Pro Gly Ala Arg 1985 1990 1995

Gin Gly Cys Met Lys Ser Ile Glu Val Lys His Ser Ala Leu Ser 2000 200S 2010

Val Glu Leu His Ser Asp Met Glu val Thr val Asn Gly Arg Leu 2015 2020 2025 val Ser val Pro Tyr val Gly Gly Asn Met Glu val Asn Val Tyr 2030 2035 2040

Gly Ala lie Met His Glu Val Arg Phe Asn His Leu Gly His lie 2045 2050 2055

Phe Thr Phe Thr Pro Gin Asn Asn Glu Phe Gin Leu Gin Leu Ser 2060 2065 2070

Pro Lys Thr Phe Ala Ser Lys Thr Tyr Gly Leu Cys Gly lie Cys 2075 2080 2085

Asp Glu Asn Gly Ala Asn Asp Phe Met Leu Arg Asp Gly Thr Val 2090 2095 2100

Thr Thr Asp Trp Lys Thr Leu Val Gin Glu Trp Thr val Gin Arg 2105 2110 2115

Pro Gly Gin Thr Cys Gin Pro lie Leu Glu Glu Gin cys Leu Val 2120 2125 2130

Pro Asp Ser Ser His Cys Gin Val Leu Leu Leu Pro Leu Phe Ala 2135 2140 2145

Glu Cys His Lys Val Leu Ala Pro Ala Thr Phe Tyr Ala lie Cys 2150 2155 2160

Gin Gin Asp Ser Cys His Gin Glu Gin val Cys Glu val lie Ala 2165 2170 2175

Ser Tyr Ala His Leu Cys Arg Thr Asn Gly val Cys val Asp Trp 2180 2185 2190

Arg Thr Pro Asp Phe Cys Ala Met ser cys Pro Pro Ser Leu val 2195 2200 2205

Tyr Asn His Cys Glu His Gly cys Pro Arg His Cys Asp Gly Asn 2210 2215 2220 val ser ser Cys Gly Asp His Pro Ser Glu Gly Cys Phe cys Pro 2225 2230 2235

Pro Asp Lys val Met Leu Glu Gly Ser Cys val Pro Glu Glu Ala 2240 2245 2250 CVS Thr Gin Cys ile Gly Glu Asp Gly Val Gin His Gin Phe Leu 2255 2260 2265

Glu Ala Trp val Pro Asp His Gin Pro Cys Gin lie Cys Thr Cys 2270 2275 2280

Leu Ser Gly Arg Lys val Asn Cys Thr Thr Gin pro Cys pro Thr 2285 2290 2295

Ala Lys Ala Pro Thr cys Gly Leu Cys Glu Val Ala Arg Leu Arg 2300 2305 2310

Gin Asn Ala Asp Gin cys Cys Pro Glu Tyr Glu Cys val cys Asp 2315 2320 2325 pro Val Ser Cys Asp Leu Pro Pro val Pro His Cys Glu Arg Gly 2330 2335 2340

Leu Gin Pro Thr Leu Thr Asn Pro Gly Glu Cys Arg Pro Asn Phe 2345 2350 2355

Thr Cys Ala Cys Arg Lys Glu Glu cys Lys Arg Val Ser Pro Pro 2360 2365 2370 ser Cys Pro Pro His Arg Leu Pro Thr Leu Arg Lys Thr Gin Cys 2375 2380 2385

Cys Asp Glu Tyr Glu Cys Ala Cys Asn cys val Asn Ser Thr Val 2390 2395 2400

Ser Cys Pro Leu Gly Tyr Leu Ala Ser Thr Ala Thr Asn Asp Cys 2405 2410 2415

Gly Cys Thr Thr Thr Thr cys Leu Pro Asp Lys val Cys val His 2420 2425 2430

Arg Ser Thr lie Tyr Pro val Gly Gin Phe Trp Glu Glu Gly cys 2435 2440 2445

Asp Val Cys Thr Cys Thr Asp Met Glu Asp Ala val Met Gly Leu 2450 2455 2460

Arg Val Ala Gin Cys ser Gin Lys Pro Cys Glu Asp Ser cys Arg 2465 2470 2475

Ser Gly Phe Thr Tyr val Leu His Glu Gly Glu cys Cys Gly Arg 2480 2485 2490

Cys Leu Pro Ser Ala Cys Glu val val Thr Gly Ser pro Arg Gly 2495 2500 2505

Asp ser Gin Ser ser Trp Lys ser val Gly Ser Gin Trp Ala Ser 2510 2515 2520 pro Glu Asn Pro cys Leu ile Asn Glu cys val Arg Val Lys Glu 2525 2530 2535

Glu Val Phe Ile Gin Gin Arg Asn Val Ser cys Pro Gin Leu Glu 2540 2545 2550 val Pro val Cys Pro Ser Gly Phe Gin Leu Ser cys Lys Thr Ser 2555 2560 2565

Ala Cys Cys Pro Ser Cys Arg Cys Glu Arg Met Glu Ala cys Met 2570 2575 2580

Leu Asn Gly Thr val ile Gly Pro Gly Lys Thr val Met ile Asp 2585 2590 2595 val Cys Thr Thr Cys Arg cys Met val Gin val Gly val Ile Ser 2600 2605 2610

Gly Phe Lys Leu Glu Cys Arg Lys Thr Thr Cys Asn Pro Cys Pro 2615 2620 2625

Leu Gly Tyr Lys Glu Glu Asn Asn Thr Gly Glu Cys Cys Gly Arg 2630 2635 2640

Cys Leu Pro Thr Ala Cys Thr ile Gin Leu Arg Gly Gly Gin Ile 2645 2650 2655

Met Thr Leu Lys Arg Asp Glu Thr Leu Gin Asp Gly Cys Asp Thr 2660 2665 2670

His Phe cys Lys val Asn Glu Arg Gly Glu Tyr Phe Trp Glu Lys 2675 2680 J 2685

Arg val Thr Gly cys Pro Pro Phe Asp Glu His Lys cys Leu Ala 2690 2695 2700

Glu Gly Gly Lys ile Met Lys ile Pro Gly Thr Cys Cys Asp Thr 2705 2710 2715 cys Glu Glu Pro Glu Cys Asn Asp ile Thr Ala Arg Leu Gin Tyr 2720 2725 2730

Val Lys val Gly Ser cys Lys ser Glu val Glu Val Asp ile His 2735 2740 2745

Tyr cys Gin Gly Lys Cys Ala ser Lys Ala Met Tyr Ser ile Asp 2750 2755 2760 ile Asn Asp val Gin Asp Gin cys Ser Cys Cys Ser Pro Thr Arg 2765 2770 u/s

Thr Glu Pro Met Gin val Ala Leu His Cys Thr Asn Gly Ser val

2780 2785 Z/aU val Tyr His Glu val Leu Asn Ala Met Glu Cys Lys cys ser Pro 2795 2800 Zoln

Arg Lys cys ser Lys ser Ser Gly Gly ser Gly Gly Ser Gly Gly 2810 2815 2820 ser Gly Gly ser Gly Gly ser Gly Gly Ser Gly Gly ser Gly Gly 2825 2830 2835

Ser Gly Gly Ser Gly Ser Asp Ala His Lys Ser Glu val Ala His 2840 2845 2850

Ara Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu val Leu 2855 2860 2865 ile Ala Phe Ala Gin Tyr Leu Gin Gin cys Pro Phe Glu Asp His 2870 2875 2880 val Lys Leu val Asn Glu val Thr Glu Phe Ala Lys Thr cys val 2885 2890 2895

Ala Asp Glu Ser Ala Glu Asn cys Asp Lys Ser Leu His Thr Leu 2900 2905 2910

Phe Gly Asp Lys Leu Cys Thr val Ala Thr Leu Arg Glu Thr Tyr 2915 2920 2925

Gly Glu Met Ala Asp Cys Cys Ala Lys Gin Glu Pro Glu Arg Asn 2930 2935 2940

Glu cys Phe Leu Gin His Lys Asp Asp Asn Pro Asn Leu Pro Arg 2945 2950 2955

Leu yaL Arg Pro Glu Val AsP val Met cys Thr Ala Phe His Asp 2960 2965 2970

Asn Glu Thr Phe Leu Lys Tyr Leu Tyr Glu ile Ala Arg 2975 2980 2985

Arg Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys zyau 2995 3000 ΑΓ9 3005 LyS Ala Ala Phe 3010 GlU Cys Cys Gln Ala Ala Asp Lys

Ala Ala Cys Leu Leu pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 3020 3025 3030

Lys Ala Ser ser Ala Lys Gin Arg Leu Lys cys Ala Ser Leu Gin 3035 3040 3045

Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala val Ala Arg Leu 3050 3055 3060

Ser Gin Arg Phe Pro Lys Ala Glu Phe Ala Glu val Ser Lys Leu 3065 3070 3075 val Thr Asp Leu Thr Lys val His Thr Glu Cys cys His Gly Asp 3080 3085 3090

Leu Leu Glu cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr lie 3095 3100 3105

Cys Glu Asn Gin Asp Ser lie Ser Ser Lys Leu Lys Glu Cys Cys 3110 3115 3120

Glu Lys Pro Leu Leu Glu Lys Ser His Cys lie Ala Glu val Glu 3125 3130 3135

Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 3140 3145 3150 val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp 3155 3160 3165

Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro 3170 3175 3180

Asp Tyr ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu 3185 3190 3195

Thr Thr Leu Glu Lys Cys cys Ala Ala Ala Asp Pro His Glu Cys 3200 3205 3210

Tyr Ala Lys val Phe Asp Glu Phe Lys Pro Leu val Glu Glu Pro 3215 3220 3225

Gin Asn Leu lie Lys Gin Asn cys Glu Leu Phe Glu Gin Leu Gly 3230 3235 3240

Glu Tyr Lys Phe Gin Asn Ala Leu Leu val Arg Tyr Thr Lys Lys 3245 3250 3255 val Pro Gin Val ser Thr Pro Thr Leu val Glu val Ser Arg Asn 3260 3265 3270

Leu Gly Lys val Gly ser Lys Cys Cys Lys His Pro Glu Ala Lys 3275 3280 3285

Arg Met Pro cys Ala Glu Asp Tyr Leu Ser val val Leu Asn Gin 3290 3295 3300

Leu Cys Val Leu His Glu Lys Thr pro Val Ser Asp Arg Val Thr 3305 3310 3315

Lys Cys Cys Thr Glu Ser Leu val Asn Arg Arg Pro Cys Phe ser 3320 3325 3330

Ala Leu Glu val Asp Glu Thr Tyr val Pro Lys Glu Phe Asn Ala 3335 3340 3345

Glu Thr Phe Thr Phe His Ala Asp ile Cys Thr Leu Ser Glu Lys 3350 3355 3360

Glu Arg Gin ile Lys Lys Gin Thr Ala Leu Val Glu Leu val Lys 3365 3370 3375

His Lys pro Lys Ala Thr Lys Glu Gin Leu Lys Ala val Met Asp 3380 3385 3390

Asp Phe Ala Ala Phe val Glu Lys Cys cys Lys Ala Asp Asp Lys 3395 3400 3405

Glu Thr cys Phe Ala Glu Glu Gly Lys Lys Leu val Ala Ala Ser 3410 3415 3420

Gin Ala Ala Leu Gly Leu 3425

Claims (19)

A modified von-Willebrand Factor (VWF) or a complex comprising unmodified Factor VIII (FVIII) and modified VWF, wherein the modified VWF is fused at a C-terminal portion of the primary translation polypeptide of VWF to the N-terminal portion of a HLEP (Half Life Enhancing Ftolypeptide) wherein HLEP is albumin and wherein a. The modified VWF has a functional half-life increased by at least 25% compared to the functional half-life of the corresponding wild-type VWF, or b. The complex comprising unmodified FVIII and modified VWF has a functional half-life increased by at least 25% compared to the corresponding wild-type FVIII and wild-type VWF complex. The modified VWF or the complex comprising unmodified FVIII and modified VWF according to claim 1, wherein a. The modified VWF has an antigen half-life increased by at least 25% compared to the antigen half-life of the corresponding wild-type VWF, or b. The complex comprising unmodified FVIII and modified VWF have an antigen half-life increased by at least 25% compared to the corresponding wild-type FVIII and wild-type VWF complex. The modified VWF or the complex comprising unmodified FVIII and modified VWF according to claim 1 or 2, wherein a. The modified VWF has an increased in vivo recovery compared to the in vivo wild-type VWF refund, or b. The complex comprising unmodified FVIII and modified VWF have an increased in vivo recovery compared to the in vivo recovery of the corresponding complex comprising wild-type FVIII and wild-type VWF. The modified VWF or complex comprising unmodified FVIII and modified VWF according to claim 3, wherein the modified VWF has an in vivo refund increased by at least 10% compared to the in vivo restitution of the corresponding wild type VWF, or the complex comprising modified VWF has a in vivo restitution increased by at least 10% compared to the in vivo restitution of the corresponding wild-type FVIII and wild-type VWF complex. The modified VWF or complex comprising unmodified FVIII and modified VWF according to any one of the preceding claims, wherein the modified VWF has at least 10% of the wild-type VWF biological activity, or the complex comprising modified VWF has at least 10% of the biological activity of the corresponding complex of wild-type FVIII and wild-type VWF. The modified VWF or the complex comprising unmodified FVIII and modified VWF according to any of the preceding claims, wherein the HLEP is fused with an amino acid of VWF located at a distance from the C-terminal amino acid of up to 5% of the total length thereof. primary translation polypeptide of VWF based on the total number of amino acids in the primary translation polypeptide of VWF. The modified polypeptide or complex of claim 6, wherein 1 to 20 amino acids at the natural C-terminal of the primary translation polypeptide of VWF have been deleted and wherein the resulting C-terminal amino acid of the VWF polypeptide is fused to the N-terminal amino acid of HLEP. The modified polypeptide or complex of claim 7, wherein the natural C-terminal amino acid of the primary translation polypeptide of VWF has been deleted and wherein the resulting C-terminal amino acid of the VWF polypeptide is fused with the N-terminal amino acid of HLEP. A polynucleotide or group of polynucleotides encoding a polypeptide or complex comprising the modified VWF or a complex comprising the modified VWF according to any one of claims 1 to 8. A plasmid or vector comprising a polynucleotide according to claim 9, or a group of plasmids or vectors comprising the group of polynucleotides according to claim 9. A host cell comprising a polynucleotide or group of polynucleotides according to claim 9 or a plasmid or vector or group of plasmids or vectors according to claim 10. A method of producing a modified VWF, comprising: (a) culturing host cells according to claim 11 under conditions such that the modified VWF is expressed, and (b) optionally recovering the modified VWF from the host cells or from the culture medium. A pharmaceutical composition comprising a modified VWF or a complex comprising the modified VWF of any one of claims 1 to 8, a polynucleotide or group of polynucleotides according to claim 9, or a plasmid or vector or group of plasmids or vectors according to claim 10th Use of a VWF or complex comprising the modified VWF of any one of claims 1 to 8, a polynucleotide or group of polynucleotides of claim 9, or a plasmid or vector or group of plasmids or vectors of claim 10, or of a host cell according to claim 11 for the manufacture of a medicament for treating or preventing a blood coagulation disorder. Use according to claim 14, wherein the blood coagulation disorder is haemophilia A. Use according to claim 14, wherein the blood coagulation disorder is von Willebrand's disease. Use according to claim 15 or 16, wherein the treatment comprises human gene therapy. A method of producing a modified VWF with increased functional half-life, comprising fusing the N-terminal portion of a half-time-enhancing polypeptide with a C-terminal portion of the primary translation polypeptide of VWF. A process for preparing a complex comprising unmodified VWF and modified VWF by mixing wild-type FVIII with a modified VWF prepared by the method of claim 18.